Nmda receptor modulators and uses thereof

ABSTRACT

Disclosed are compounds having enhanced potency in the modulation of NMDA receptor activity. Such compounds are contemplated for use in the treatment of diseases and disorders, such as learning, cognitive activities, and analgesia, particularly in alleviating and/or reducing neuropathic pain. Orally available formulations and other pharmaceutically acceptable delivery forms of the compounds, including intravenous formulations, are also disclosed.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 13/525,861, filed Jun.18, 2012, which claims priority to U.S. Provisional Application No.61/550,782, filed Oct. 24, 2011, and is a continuation in part of PCT/US11/24583, filed Feb. 11, 2011, claiming priority to U.S. ProvisionalApplication No. 61/303,472, filed Feb. 11, 2010; all of which are herebyincorporated by reference in their entireties.

BACKGROUND

An N-methyl-d-aspartate (NMDA) receptor is a postsynaptic, ionotropicreceptor that is responsive to, inter alia, the excitatory amino acidsglutamate and glycine and the synthetic compound NMDA. The NMDA receptorcontrols the flow of both divalent and monovalent ions into thepostsynaptic neural cell through a receptor associated channel (Fosteret al., Nature 1987, 329:395-396; Mayer et al., Trends in Pharmacol.Sci. 1990, 11:254-260). The NMDA receptor has been implicated duringdevelopment in specifying neuronal architecture and synapticconnectivity, and may be involved in experience-dependent synapticmodifications. In addition, NMDA receptors are also thought to beinvolved in long term potentiation and central nervous system disorders.

The NMDA receptor plays a major role in the synaptic plasticity thatunderlies many higher cognitive functions, such as memory acquisition,retention and learning, as well as in certain cognitive pathways and inthe perception of pain (Collingridge et al., The NMDA Receptor, OxfordUniversity Press, 1994). In addition, certain properties of NMDAreceptors suggest that they may be involved in theinformation-processing in the brain that underlies consciousness itself.

The NMDA receptor has drawn particular interest since it appears to beinvolved in a broad spectrum of CNS disorders. For instance, duringbrain ischemia caused by stroke or traumatic injury, excessive amountsof the excitatory amino acid glutamate are released from damaged oroxygen deprived neurons. This excess glutamate binds to the NMDAreceptors which opens their ligand-gated ion channels; in turn thecalcium influx produces a high level of intracellular calcium whichactivates a biochemical cascade resulting in protein degradation andcell death. This phenomenon, known as excitotoxicity, is also thought tobe responsible for the neurological damage associated with otherdisorders ranging from hypoglycemia and cardiac arrest to epilepsy. Inaddition, there are preliminary reports indicating similar involvementin the chronic neurodegeneration of Huntington's, Parkinson's, andAlzheimer's diseases. Activation of the NMDA receptor has been shown tobe responsible for post-stroke convulsions, and, in certain models ofepilepsy, activation of the NMDA receptor has been shown to be necessaryfor the generation of seizures. Neuropsychiatric involvement of the NMDAreceptor has also been recognized since blockage of the NMDA receptorCa⁺⁺ channel by the animal anesthetic PCP (phencyclidine) produces apsychotic state in humans similar to schizophrenia (reviewed in Johnson,K. and Jones, S., 1990). Further, NMDA receptors have also beenimplicated in certain types of spatial learning.

The NMDA receptor is believed to consist of several protein chainsembedded in the postsynaptic membrane. The first two types of subunitsdiscovered so far form a large extracellular region, which probablycontains most of the allosteric binding sites, several transmembraneregions looped and folded so as to form a pore or channel, which ispermeable to Ca⁺⁺, and a carboxyl terminal region. The opening andclosing of the channel is regulated by the binding of various ligands todomains (allosteric sites) of the protein residing on the extracellularsurface. The binding of the ligands is thought to affect aconformational change in the overall structure of the protein which isultimately reflected in the channel opening, partially opening,partially closing, or closing.

NMDA receptor compounds may exert dual (agonist/antagonist) effect onthe NMDA receptor through the allosteric sites. These compounds aretypically termed “partial agonists”. In the presence of the principalsite ligand, a partial agonist will displace some of the ligand and thusdecrease Ca⁺⁺ flow through the receptor. In the absence of or loweredlevel of the principal site ligand, the partial agonist acts to increaseCa⁺⁺ flow through the receptor channel.

A need continues to exist in the art for novel and more specific/potentcompounds that are capable of binding the glycine binding site of NMDAreceptors, and provide pharmaceutical benefits. In addition, a needcontinues to exist in the medical arts for an orally deliverable formsof such compounds.

SUMMARY

Provided herein, at least in part, are compounds that are NMDAmodulators, for example, partial agonists of NMDA. For example,disclosed herein are compounds represented by the formula: A compoundrepresented by:

wherein:and pharmaceutically acceptable salts, stereoisomers, metabolites, andhydrates thereof, wherein: R¹, R², R³, R⁴, and X are as defined below.

Also provided herein are pharmaceutically acceptable compositionscomprising a disclosed compound, and a pharmaceutically acceptableexcipient. For example, such compositions may be suitable for oraladministration to a patient.

In another aspect, a method of treating a condition selected from thegroup consisting of depression, Alzheimer's disease, memory loss thataccompanies early stage Alzheimer's disease, attention deficit disorder,ADHD, schizophrenia, anxiety, in a patient in need thereof is provided.The method comprises administering to the patient a pharmaceuticallyeffective amount of a disclosed compound and pharmaceutically acceptablesalts, stereoisomers, metabolites, and hydrates thereof.

DETAILED DESCRIPTION

This disclosure is generally directed to compounds that are capable ofmodulating NMDA, e.g., NMDA antagonists or partial agonists, andcompositions and/or methods of using the disclosed compounds.

Definitions

In some embodiments, the compounds, as described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas, refer tothe replacement of hydrogen radicals in a given structure with theradical of a specified substituent.

In some instances, when more than one position in any given structuremay be substituted with more than one substituent selected from aspecified group, the substituent may be either the same or different atevery position.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. In some embodiments, heteroatoms suchas nitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalencies of the heteroatoms. Non-limiting examples of substituentsinclude acyl; aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; cycloalkoxy; heterocyclylalkoxy;heterocyclyloxy; heterocyclyloxyalkyl; alkenyloxy; alkynyloxy; aryloxy;heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroarylthio; oxo;—F; —Cl; —Br; —I; —OH; —NO₂; —N₃; —CN; —SCN; —SR^(x); —CF₃; —CH₂CF₃;—CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —OR^(x), —C(O)R^(x);—CO₂(R^(x)); —C(O)N(R^(x))₂; —C(NR^(x))N(R^(x))₂; —OC(O)R^(x);—OCO₂R^(x); —OC(O)N(R^(x))₂; —N(R^(x))₂; —SOR^(x); —S(O)₂R^(x);—NR^(x)C(O)R^(x); —NR^(x)C(O)N(R^(x))₂; —NR^(x)C(O)OR^(x);—NR^(x)C(NR^(x))N(R^(x))₂; and —C(R^(x))₃; wherein each occurrence ofR^(x) independently includes, but is not limited to, hydrogen, halogen,acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Furthermore, the compounds described herein are not intended to belimited in any manner by the permissible substituents of organiccompounds. In some embodiments, combinations of substituents andvariables described herein may be preferably those that result in theformation of stable compounds. The term “stable,” as used herein, refersto compounds which possess stability sufficient to allow manufacture andwhich maintain the integrity of the compound for a sufficient period oftime to be detected and preferably for a sufficient period of time to beuseful for the purposes detailed herein.

The terms “aryl” and “heteroaryl,” as used herein, refer to mono- orpolycyclic unsaturated moieties having preferably 3-14 carbon atoms,each of which may be substituted or unsubstituted. In certainembodiments, “aryl” refers to a mono- or bicyclic carbocyclic ringsystem having one or two aromatic rings including, but not limited to,phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. Incertain embodiments, “heteroaryl” refers to a mono- or bicyclicheterocyclic ring system having one or two aromatic rings in which one,two, or three ring atoms are heteroatoms independently selected from thegroup consisting of S, O, and N and the remaining ring atoms are carbon.Non-limiting examples of heteroaryl groups include pyridyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, and the like.

The term “alkyl” as used herein refers to a saturated straight orbranched hydrocarbon, for example, such as a straight or branched groupof 1-6, 1-4, or 1-3 carbon atom, referred to herein as C₁-C₆alkyl,C₁-C₄alkyl, and C₁-C₃alkyl, respectively. Exemplary alkyl groupsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl,3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl,2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl,2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl,isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl,etc.

Alkyl, alkenyl and alkynyl groups can optionally be substituted, if notindicated otherwise, with one or more groups selected from alkoxy,alkyl, cycloalkyl, amino, halogen, and —C(O)alkyl. In certainembodiments, the alkyl, alkenyl, and alkynyl groups are not substituted,i.e., they are unsubstituted.

The term “amine” or “amino” as used herein refers to a radical of theform —NR^(d)R^(e), where R^(d) and R^(e) are independently selected fromhydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,haloalkyl, heteroaryl, and heterocyclyl. The amino also may be cyclic,for example, R^(d) and R^(e) are joined together with the N to form a 3-to 12-membered ring, e.g., morpholino or piperidinyl. The term aminoalso includes the corresponding quaternary ammonium salt of any aminogroup, e.g., —[N(R^(d))(R^(e))(R^(f))]+. Exemplary amino groups includeaminoalkyl groups, wherein at least one of R^(d), R^(e), or R^(f) is analkyl group. In certain embodiment, R^(d) and R^(e) are hydrogen oralkyl.

The terms “halo” or “halogen” or “Hal” as used herein refer to F, Cl,Br, or I. The term “haloalkyl” as used herein refers to an alkyl groupsubstituted with one or more halogen atoms.

The terms “heterocyclyl” or “heterocyclic group” are art-recognized andrefer to saturated or partially unsaturated 3- to 10-membered ringstructures, alternatively 3- to 7-membered rings, whose ring structuresinclude one to four heteroatoms, such as nitrogen, oxygen, and sulfur.Heterocycles may also be mono-, bi-, or other multi-cyclic ring systems.A heterocycle may be fused to one or more aryl, partially unsaturated,or saturated rings. Heterocyclyl groups include, for example, biotinyl,chromenyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl,dithiazolyl, homopiperidinyl, imidazolidinyl, isoquinolyl,isothiazolidinyl, isoxazolidinyl, morpholinyl, oxolanyl, oxazolidinyl,phenoxanthenyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl,pyrazolinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolidin-2-onyl,pyrrolinyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl,tetrahydroquinolyl, thiazolidinyl, thiolanyl, thiomorpholinyl,thiopyranyl, xanthenyl, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringmay be substituted at one or more positions with substituents such asalkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amido, amidino, amino, aryl,arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl,ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl,hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato,sulfate, sulfide, sulfonamido, sulfonyl and thiocarbonyl. In certainembodiments, the heterocyclic group is not substituted, i.e., theheterocyclic group is unsubstituted.

The terms “hydroxy” and “hydroxyl” as used herein refers to the radical—OH.

The term “oxo” as used herein refers to the radical ═O.

“Pharmaceutically or pharmacologically acceptable” include molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. “For human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

As used in the present disclosure, the term “partial NMDA receptoragonist” is defined as a compound that is capable of binding to aglycine binding site of an NMDA receptor; at low concentrations a NMDAreceptor agonist acts substantially as agonist and at highconcentrations it acts substantially as an antagonist. Theseconcentrations are experimentally determined for each and every “partialagonist.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. In one embodiment, the carrieris suitable for parenteral administration. Alternatively, the carriercan be suitable for intravenous, intraperitoneal, intramuscular,sublingual or oral administration. Pharmaceutically acceptable carriersinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The term “pharmaceutically acceptable salt(s)” as used herein refers tosalts of acidic or basic groups that may be present in compounds used inthe present compositions. Compounds included in the present compositionsthat are basic in nature are capable of forming a wide variety of saltswith various inorganic and organic acids. The acids that may be used toprepare pharmaceutically acceptable acid addition salts of such basiccompounds are those that form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, including but notlimited to malate, oxalate, chloride, bromide, iodide, nitrate, sulfate,bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate,salicylate, citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.Compounds included in the present compositions that include an aminomoiety may form pharmaceutically acceptable salts with various aminoacids, in addition to the acids mentioned above. Compounds included inthe present compositions that are acidic in nature are capable offorming base salts with various pharmacologically acceptable cations.Examples of such salts include alkali metal or alkaline earth metalsalts and, particularly, calcium, magnesium, sodium, lithium, zinc,potassium, and iron salts.

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asgeometric isomers, enantiomers or diastereomers. The term“stereoisomers” when used herein consist of all geometric isomers,enantiomers or diastereomers. These compounds may be designated by thesymbols “R” or “S,” depending on the configuration of substituentsaround the stereogenic carbon atom. The present invention encompassesvarious stereoisomers of these compounds and mixtures thereof.Stereoisomers include enantiomers and diastereomers. Mixtures ofenantiomers or diastereomers may be designated “(±)” in nomenclature,but the skilled artisan will recognize that a structure may denote achiral center implicitly.

Individual stereoisomers of compounds of the present invention can beprepared synthetically from commercially available starting materialsthat contain asymmetric or stereogenic centers, or by preparation ofracemic mixtures followed by resolution methods well known to those ofordinary skill in the art. These methods of resolution are exemplifiedby (1) attachment of a mixture of enantiomers to a chiral auxiliary,separation of the resulting mixture of diastereomers byrecrystallization or chromatography and liberation of the optically pureproduct from the auxiliary, (2) salt formation employing an opticallyactive resolving agent, or (3) direct separation of the mixture ofoptical enantiomers on chiral chromatographic columns. Stereoisomericmixtures can also be resolved into their component stereoisomers by wellknown methods, such as chiral-phase gas chromatography, chiral-phasehigh performance liquid chromatography, crystallizing the compound as achiral salt complex, or crystallizing the compound in a chiral solvent.Stereoisomers can also be obtained from stereomerically-pureintermediates, reagents, and catalysts by well known asymmetricsynthetic methods.

Geometric isomers can also exist in the compounds of the presentinvention. The symbol

denotes a bond that may be a single, double or triple bond as describedherein. The present invention encompasses the various geometric isomersand mixtures thereof resulting from the arrangement of substituentsaround a carbon-carbon double bond or arrangement of substituents arounda carbocyclic ring. Substituents around a carbon-carbon double bond aredesignated as being in the “Z” or “E” configuration wherein the terms“Z” and “E” are used in accordance with IUPAC standards. Unlessotherwise specified, structures depicting double bonds encompass boththe “E” and “Z” isomers.

Substituents around a carbon-carbon double bond alternatively can bereferred to as “cis” or “trans,” where “cis” represents substituents onthe same side of the double bond and “trans” represents substituents onopposite sides of the double bond. The arrangement of substituentsaround a carbocyclic ring are designated as “cis” or “trans.” The term“cis” represents substituents on the same side of the plane of the ringand the term “trans” represents substituents on opposite sides of theplane of the ring. Mixtures of compounds wherein the substituents aredisposed on both the same and opposite sides of plane of the ring aredesignated “cis/trans.”

The compounds disclosed herein can exist in solvated as well asunsolvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms. In one embodiment, thecompound is amorphous. In one embodiment, the compound is a polymorph.In another embodiment, the compound is in a crystalline form.

The invention also embraces isotopically labeled compounds of theinvention which are identical to those recited herein, except that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorus, fluorine and chlorine, such as ²H, ³H, ¹³C, ⁴C, ¹⁵N, ¹⁸O,¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively.

Certain isotopically-labeled disclosed compounds (e.g., those labeledwith ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C)isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Isotopically labeled compounds of the invention cangenerally be prepared by following procedures analogous to thosedisclosed in the e.g., Examples herein by substituting an isotopicallylabeled reagent for a non-isotopically labeled reagent.

As used in the present disclosure, “NMDA” is defined asN-methyl-d-aspartate.

In the present specification, the term “therapeutically effectiveamount” means the amount of the subject compound that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician. The compounds of the invention are administered intherapeutically effective amounts to treat a disease. Alternatively, atherapeutically effective amount of a compound is the quantity requiredto achieve a desired therapeutic and/or prophylactic effect, such as anamount which results in defined as that amount needed to give maximalenhancement of a behavior (for example, learning), physiologicalresponse (for example, LTP induction), or inhibition of neuropathicpain.

Compounds

Disclosed compounds include those represented by the formula:

and pharmaceutically acceptable salts, stereoisomers, metabolites, andhydrates thereof, wherein:

R¹, R², R³, and R⁴ may be independently selected from the groupconsisting of hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted aryl; substituted or unsubstitutedheteroaryl; —OR^(x); —NO₂; —N₃; —CN; —SCN; —SR^(x); —C(O)R^(x);—CO₂(R^(x)); —C(O)N(R^(x))₂; —C(NR^(x))N(R^(x))₂; —OC(O)R^(x);—OCO₂R^(x); —OC(O)N(R^(x))₂; —N(R^(x))₂; —SOR^(x); —S(O)₂R^(x);—NR^(x)C(O)R^(x); —NR^(x)C(O)N(R^(x))₂; —NR^(x)C(O)OR^(x);—NR^(x)C(NR^(x))N(R^(x))₂; and —C(R^(x))₃; wherein each occurrence ofR^(x) is independently selected from the group consisting of hydrogen;halogen; acyl; optionally substituted aliphatic; optionally substitutedheteroaliphatic; optionally substituted aryl; and optionally substitutedheteroaryl;

R⁵ and R⁶ may be independently selected from the group consisting of-Q-Ar and hydrogen, provided that at least one of R⁵ and R⁶ is -Q-Ar;wherein Q is independently selected from the group consisting of cyclicor acyclic, substituted or unsubstituted, branched or unbranchedaliphatic; cyclic or acyclic, substituted or unsubstituted, branched orunbranched heteroaliphatic; and a bond; and wherein Ar is selected fromthe group consisting substituted or unsubstituted aryl, and substitutedor unsubstituted heteroaryl; or R⁵ and R⁶, together with the atoms towhich they are attached, form a substituted or unsubstituted 4-6membered heterocyclic or cycloalkyl ring;

R⁷ and R⁸ may be independently selected from the group consisting ofhydrogen; halogen; hydroxyl; substituted or unsubstituted C₁-C₆ alkyl;substituted or unsubstituted C₁-C₆ alkoxy; and substituted orunsubstituted aryl; or R⁷ and R⁸, together with the atoms to which theyare attached, form a substituted or unsubstituted 4-6 memberedheterocyclic or cycloalkyl ring;

R⁹ and R¹⁰ may be independently selected from the group consisting ofhydrogen; C₁-C₆ alkyl, optionally substituted by one or moresubstituents each independently selected from the group consisting ofhalogen, oxo, and hydroxyl; C₂₋₆alkenyl, optionally substituted by oneor more substituents each independently selected from the groupconsisting of halogen, oxo, and hydroxyl; C₂₋₆alkynyl, optionallysubstituted by one or more substituents each independently selected fromthe group consisting of halogen, oxo, and hydroxyl; C₃₋₆cycloalkyl,optionally substituted by one or more substituents each independentlyselected from the group consisting of C₁₋₆alkyl, halogen, oxo, andhydroxyl; phenyl, optionally substituted by one or more substituentseach independently selected from the group consisting of C₁₋₆alkyl;C₁₋₆alkoxy; halogen; hydroxyl; —C(O)R^(x); —CO₂(R^(x)); —C(O)N(R^(x))₂;—C(NR^(x))N(R^(x))₂; and —C(R^(x))₃;

X is selected from the group consisting of OR^(x) or NR^(x)R^(x);wherein each occurrence of R^(x) is independently selected from thegroup consisting of hydrogen; halogen; C₁₋₆alkyl; C₂₋₆alkenyl;C₂₋₆alkynyl; C₃₋₆cycloalkyl; and phenyl; or R⁹ and R¹⁰, together with N,form a 4-6 membered heterocyclic ring, optionally substituted by one ormore substituents each independently selected from the group consistingof C₁₋₆alkyl, halogen, oxo, and hydroxyl.

In some embodiments, R¹, R², R³, and R⁴ may be independently selectedfrom the group consisting of hydrogen; halogen; C₁₋₆alkyl; C₂₋₆alkenyl;C₂₋₆alkynyl; C₃₋₆cycloalkyl; phenyl; naphthyl; heteroaryl; heterocyclyl;C₃₋₆cycloalkyl-C₁₋₆alkyl-; phenyl-C₁₋₆alkyl-; naphthyl-C₁₋₆alkyl-;heteroaryl-C₁₋₆alkyl-; and heterocyclyl-C₁₋₆alkyl-; —OR^(x); —NO₂; —N₃;—CN; —SCN; —SR^(x); —C(O)R^(x); —CO₂(R^(x)); —C(O)N(R^(x))₂;—C(NR^(x))N(R^(x))₂; —OC(O)R^(x); —OCO₂R^(x); —OC(O)N(R^(x))₂;—N(R^(x))₂; —SOR^(x); —S(O)₂R^(x); —NR^(x)C(O)R^(x);—NR^(x)C(O)N(R^(x))₂; —NR^(x)C(O)OR^(x); —NR^(x)C(NR^(x))N(R^(x))₂; and—C(R^(x))₃; wherein heteroaryl is a 5-6 membered ring having one, two,or three heteroatoms each independently selected from N, O, or S;wherein heteroaryl is optionally substituted with one or moresubstituents each independently selected from R^(b); whereinheterocyclyl is a 4-7 membered ring optionally substituted by one ormore substituents each independently selected from R^(c); wherein whenheterocyclyl contains a —NH— moiety, that —NH— moiety is optionallysubstituted by R^(d); wherein C₂₋₆alkenyl and C₂₋₆alkynyl, are eachindependently optionally substituted by one or more substituents eachindependently selected from R^(e); wherein C₁₋₆alkyl is optionallysubstituted by one or more substituents each independently selected fromR^(f); wherein C₃₋₆cycloalkyl is independently optionally substituted byone or more substituents each independently selected from R^(g);

R^(b) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; C₁₋₆alkyl;C₂₋₆alkenyl; C₂₋₆alkynyl; C₃₋₆cycloalkyl; C₁₋₆alkoxy; C₃₋₆alkenyloxy;C₃₋₆alkynyloxy; C₃₋₆cycloalkoxy; C₁₋₆alkyl-S(O)_(w)—, where w is 0, 1,or 2; C₁₋₆alkylC₃₋₆cycloalkyl-; C₃₋₆cycloalkyl-C₁₋₆alkyl-;C₁₋₆alkoxycarbonyl-N(R^(a))—; C₁₋₆alkylN(R^(a))—;C₁₋₆alkyl-N(R^(a))carbonyl-; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl-;R^(a)R^(a′)N-carbonyl-N(R^(a))—; R^(a)R^(a′)N—SO₂—; andC₁₋₆alkyl-carbonyl-N(R^(a))—;

R^(a) and R^(a′) may be selected, independently for each occurrence,from the group consisting of hydrogen and C₁₋₆alkyl, or R^(a) and R^(a′)when taken together with the nitrogen to which they are attached form a4-6 membered heterocyclic ring, wherein C₁₋₆alkyl is optionallysubstituted by one or more substituents each independently selected fromthe group consisting of halogen, oxo, and hydroxyl, and wherein theheterocyclic ring is optionally substituted by one or more substituentseach independently selected from the group consisting of halogen, alkyl,oxo, or hydroxyl;

R^(c) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; oxo; C₁₋₆alkyl;C₂₋₆alkenyl; C₂₋₆alkynyl; C₃₋₆cycloalkyl; C₁₋₆alkoxy; C₃₋₆alkenyloxy;C₃₋₆alkynyloxy; C₃₋₆cycloalkoxy; C₁₋₆alkyl-S(O)_(w)—, where w is 0, 1,or 2; C₁₋₆alkylC₃₋₆cycloalkyl-; C₃₋₆cycloalkyl-C₁₋₆alkyl-;C₁₋₆alkoxycarbonyl-N(R^(a))—; C₁₋₆alkylN(R^(a))—;C₁₋₆alkyl-N(R^(a))carbonyl-; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl-;R^(a)R^(a′)N-carbonyl-N(R^(a))—; R^(a)R^(a′)N—SO₂—; andC₁₋₆alkyl-carbonyl-N(R^(a))—;

R^(d) may be selected, independently for each occurrence, from the groupconsisting of C₁₋₆alkyl, C₁₋₆alkylcarbonyl, and C₁₋₆alkylsulfonyl,wherein C₁₋₆alkyl is optionally substituted by one or more substituentseach independently selected from halogen, hydroxyl, and R^(a)R^(a′)N—;

R^(e) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; C₁₋₄alkoxy;C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl;R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, where w is 0, 1, or 2;

R^(f) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; C₁₋₄alkoxy;C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl;R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, where w is 0, 1, or 2;

R may be selected, independently for each occurrence, from the groupconsisting of halogen, hydroxyl, —NO₂; —N₃; —CN; —SCN; C₁₋₆alkyl;C₁₋₄alkoxy; C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl;R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, where w is 0, 1, or 2;

R^(x) may be selected, independently, from the group consisting ofhydrogen; halogen; C₁₋₆alkyl; C₂₋₆alkenyl; C₂₋₆alkynyl; C₃₋₆cycloalkyl;phenyl; naphthyl; heteroaryl; heterocyclyl; C₃₋₆cycloalkyl-C₁₋₆alkyl-;phenyl-C₁₋₆alkyl-; naphthyl-C₁₋₆alkyl-; heteroaryl-C₁₋₆alkyl-; andheterocyclyl-C₁₋₆alkyl-; wherein heteroaryl is a 5-6 membered ringhaving one, two, or three heteroatoms each independently selected fromN, O, or S; wherein heteroaryl is optionally substituted with one ormore substituents each independently selected from R^(b); whereinheterocyclyl is a 4-7 membered ring optionally substituted by one ormore substituents each independently selected from R^(c); wherein whenheterocyclyl contains a —NH— moiety, that —NH— moiety is optionallysubstituted by R^(d); wherein C₂₋₆alkenyl and C₂₋₆alkynyl, are eachindependently optionally substituted by one or more substituents eachindependently selected from R^(e); wherein C₁₋₆alkyl is optionallysubstituted by one or more substituents each independently selected fromR^(f); wherein C₃₋₆cycloalkyl is independently optionally substituted byone or more substituents each independently selected from R^(g).

In certain embodiments, at least one of R¹, R², R³, and R⁴ may behydroxyl.

In some instances, at least one of R¹, R², R³, and R⁴ may be C₁-C₆alkyl, optionally substituted with one, two, or three substituentsselected independently from the group consisting of halogen, hydroxyl,—NH₂, and cyano.

In some embodiments, at least one of R⁵ and R⁶ may be —(C₁-C₆alkylene)-Ar. At least one of R⁵ and R⁶ may also be —CH₂—Ar. In somecases, at least one of R⁵ and R⁶ is -Q-phenyl. In certain examples, oneof R⁵ and R⁶ may be hydrogen.

In some cases, R⁷ and R⁸ may be independently selected from the groupconsisting of hydrogen; halogen; hydroxyl; C₁-C₆ alkyl; phenyl; andnaphthyl; or R⁷ and R⁸, together with the atoms to which they areattached, form a 4-6 membered heterocyclic or cycloalkyl ring; whereinC₁-C₆ alkyl, phenyl, naphthyl, the cycloalkyl ring, and the heterocyclicring each may be substituted independently by one or more substituentsselected from the group consisting of halogen; hydroxyl; —NO₂; —N₃; —CN;—SCN; C₁₋₄alkoxy; C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—;R^(a)R^(a′)N-carbonyl; R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, wherew is 0, 1, or 2; wherein R^(a) and R^(a′) may be selected, independentlyfor each occurrence, from the group consisting of hydrogen andC₁₋₆alkyl, or R^(a) and R^(a′) when taken together with the nitrogen towhich they are attached form a 4-6 membered heterocyclic ring, whereinC₁₋₆alkyl is optionally substituted by one or more substituents eachindependently selected from the group consisting of halogen, oxo, andhydroxyl, and wherein the heterocyclic ring is optionally substituted byone or more substituents each independently selected from the groupconsisting of halogen, alkyl, oxo, or hydroxyl.

In some cases, R⁷ and R⁸ may be hydrogen.

X may be, for example, selected from the group consisting of OH and NH₂.

In an exemplary embodiment, a compound may be represented by:

wherein X is OH or NH₂.

In an exemplary embodiment, a compound may be represented by:

In another exemplary embodiment, a compound may be represented by:

In yet another exemplary embodiment, a compound may be represented by:

Provided herein, for example, is a compound represented by:

wherein X is OH or NH₂, and pharmaceutically acceptable salts thereof.

Disclosed compounds also include those represented by the formula:

and pharmaceutically acceptable salts, stereoisomers, metabolites, andhydrates thereof,wherein:

R¹ and R³ may be independently selected from the group consisting ofhydrogen; halogen; cyclic or acyclic, substituted or unsubstituted,branched or unbranched aliphatic; cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; —OR^(x);—NO₂; —N₃; —CN; —SCN; —SR^(x); —C(O)R^(x); —CO₂(R^(x)); —C(O)N(R^(x))₂;—C(NR^(x))N(R^(x))₂; —OC(O)R^(x); —OCO₂R^(x); —OC(O)N(R^(x))₂;—N(R^(x))₂; —SOR^(x); —S(O)₂R^(x); —NR^(x)C(O)R^(x);—NR^(x)C(O)N(R^(x))₂; —NR^(x)C(O)OR^(x); —NR^(x)C(NR^(x))N(R^(x))₂; and—C(R^(x))₃; wherein each occurrence of R^(x) is independently selectedfrom the group consisting of hydrogen; halogen; acyl; optionallysubstituted aliphatic; optionally substituted heteroaliphatic;optionally substituted aryl; and optionally substituted heteroaryl;

R² and R⁴ may be independently selected from the group consisting ofhydrogen and —OR^(x), provided that at least one of R² and R⁴ ishydrogen, wherein R^(x) is selected from the group consisting ofhydrogen; halogen; acyl; optionally substituted aliphatic; optionallysubstituted heteroaliphatic; optionally substituted aryl; and optionallysubstituted heteroaryl;

R⁵ and R⁶ may be independently selected from the group consisting of-Q-Ar and hydrogen; wherein Q is independently selected from the groupconsisting of cyclic or acyclic, substituted or unsubstituted, branchedor unbranched aliphatic; cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic; and a bond; andwherein Ar is selected from the group consisting substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl; or R⁵and R⁶, together with the atoms to which they are attached, form asubstituted or unsubstituted 4-6 membered heterocyclic or cycloalkylring;

R⁷ and R⁸ are independently selected from the group consisting ofhydrogen; halogen; hydroxyl; substituted or unsubstituted C₁-C₆ alkyl;substituted or unsubstituted C₁-C₆ alkoxy; and substituted orunsubstituted aryl; or R⁷ and R⁸, together with the atoms to which theyare attached, form a substituted or unsubstituted 4-6 memberedheterocyclic or cycloalkyl ring;

R⁹ and R¹⁰ may be independently selected from the group consisting ofhydrogen; C₁-C₆ alkyl, optionally substituted by one or moresubstituents each independently selected from the group consisting ofhalogen, oxo, and hydroxyl; C₂₋₆alkenyl, optionally substituted by oneor more substituents each independently selected from the groupconsisting of halogen, oxo, and hydroxyl; C₂₋₆alkynyl, optionallysubstituted by one or more substituents each independently selected fromthe group consisting of halogen, oxo, and hydroxyl; C₃₋₆cycloalkyl,optionally substituted by one or more substituents each independentlyselected from the group consisting of C₁₋₆alkyl, halogen, oxo, andhydroxyl; phenyl, optionally substituted by one or more substituentseach independently selected from the group consisting of C₁₋₆alkyl;C₁₋₆alkoxy; halogen; hydroxyl; —C(O)R^(x); —CO₂(R^(x)); —C(O)N(R^(x))₂;—C(NR^(x))N(R^(x))₂; and —C(R^(x))₃; wherein each occurrence of R^(x) isindependently selected from the group consisting of hydrogen; halogen;C₁₋₆alkyl; C₂₋₆alkenyl; C₂₋₆alkynyl; C₃₋₆cycloalkyl; and phenyl; or R⁹and R¹⁰, together with N, form a 4-6 membered heterocyclic ring,optionally substituted by one or more substituents each independentlyselected from the group consisting of C₁₋₆alkyl, halogen, oxo, andhydroxyl.

In some embodiments, R¹ and R³ may be independently selected from thegroup consisting of hydrogen; halogen; C₁₋₆alkyl; C₂₋₆alkenyl;C₂₋₆alkynyl; C₃₋₆cycloalkyl; phenyl; naphthyl; heteroaryl; heterocyclyl;C₃₋₆cycloalkyl-C₁₋₆alkyl-; phenyl-C₁₋₆alkyl-; naphthyl-C₁₋₆alkyl-;heteroaryl-C₁₋₆alkyl-; and heterocyclyl-C₁₋₆alkyl-; —OR^(x); —NO₂; —N₃;—CN; —SCN; —SR^(x); —C(O)R^(x); —CO₂(R^(x)); —C(O)N(R^(x))₂;—C(NR^(x))N(R^(x))₂; —OC(O)R^(x); —OCO₂R^(x); —OC(O)N(R^(x))₂;—N(R^(x))₂; —SOR^(x); —S(O)₂R^(x); —NR^(x)C(O)R^(x);—NR^(x)C(O)N(R^(x))₂; —NR^(x)C(O)OR^(x); —NR^(x)C(NR^(x))N(R^(x))₂; and—C(R^(x))₃; wherein heteroaryl is a 5-6 membered ring having one, two,or three heteroatoms each independently selected from N, O, or S;wherein heteroaryl is optionally substituted with one or moresubstituents each independently selected from R^(b); whereinheterocyclyl is a 4-7 membered ring optionally substituted by one ormore substituents each independently selected from R^(C); wherein whenheterocyclyl contains a —NH— moiety, that —NH— moiety is optionallysubstituted by R^(d); wherein C₂₋₆alkenyl and C₂₋₆alkynyl, are eachindependently optionally substituted by one or more substituents eachindependently selected from R^(e); wherein C₁₋₆alkyl is optionallysubstituted by one or more substituents each independently selected fromR^(f); wherein C₃₋₆cycloalkyl is independently optionally substituted byone or more substituents each independently selected from R^(g);

R^(b) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; C₁₋₆alkyl;C₂₋₆alkenyl; C₂₋₆alkynyl; C₃₋₆cycloalkyl; C₁₋₆alkoxy; C₃₋₆alkenyloxy;C₃₋₆alkynyloxy; C₃₋₆cycloalkoxy; C₁₋₆alkyl-S(O)_(w)—, where w is 0, 1,or 2; C₁₋₆alkylC₃₋₆cycloalkyl-; C₃₋₆cycloalkyl-C₁₋₆alkyl-;C₁₋₆alkoxycarbonyl-N(R^(a))—; C₁₋₆alkylN(R^(a))—;C₁₋₆alkyl-N(R^(a))carbonyl-; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl-;R^(a)R^(a′)N-carbonyl-N(R^(a))—; R^(a)R^(a′)N—SO₂—; andC₁₋₆alkyl-carbonyl-N(R^(a))—;

R^(a) and R^(a′) may be selected, independently for each occurrence,from the group consisting of hydrogen and C₁₋₆alkyl, or R^(a) and R^(a′)when taken together with the nitrogen to which they are attached form a4-6 membered heterocyclic ring, wherein C₁₋₆alkyl is optionallysubstituted by one or more substituents each independently selected fromthe group consisting of halogen, oxo, and hydroxyl, and wherein theheterocyclic ring is optionally substituted by one or more substituentseach independently selected from the group consisting of halogen, alkyl,oxo, or hydroxyl;

R^(c) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; oxo; C₁₋₆alkyl;C₂₋₆alkenyl; C₂₋₆alkynyl; C₃₋₆cycloalkyl; C₁₋₆alkoxy; C₃₋₆alkenyloxy;C₃₋₆alkynyloxy; C₃₋₆cycloalkoxy; C₁₋₆alkyl-S(O)_(w)—, where w is 0, 1,or 2; C₁₋₆alkylC₃₋₆cycloalkyl-; C₃₋₆cycloalkyl-C₁₋₆alkyl-;C₁₋₆alkoxycarbonyl-N(R^(a))—; C₁₋₆alkylN(R^(a))—;C₁₋₆alkyl-N(R^(a))carbonyl-; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl-;R^(a)R^(a′)N-carbonyl-N(R^(a))—; R^(a)R^(a′)N—SO₂—; andC₁₋₆alkyl-carbonyl-N(R^(a))—;

R^(d) may be selected, independently for each occurrence, from the groupconsisting of C₁₋₆alkyl, C₁₋₆alkylcarbonyl, and C₁₋₆alkylsulfonyl,wherein C₁₋₆alkyl is optionally substituted by one or more substituentseach independently selected from halogen, hydroxyl, and R^(a)R^(a′)N—;

R^(e) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; C₁₋₄alkoxy;C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl;R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, where w is 0, 1, or 2;

R^(f) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; C₁₋₄alkoxy;C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl;R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, where w is 0, 1, or 2;

R^(g) may be selected, independently for each occurrence, from the groupconsisting of halogen, hydroxyl, —NO₂; —N₃; —CN; —SCN; C₁₋₆alkyl;C₁₋₄alkoxy; C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl;R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, where w is 0, 1, or 2;

R^(x) may be selected, independently, from the group consisting ofhydrogen; halogen; C₁₋₆alkyl; C₂₋₆alkenyl; C₂₋₆alkynyl; C₃₋₆cycloalkyl;phenyl; naphthyl; heteroaryl; heterocyclyl; C₃₋₆cycloalkyl-C₁₋₆alkyl-;phenyl-C₁₋₆alkyl-; naphthyl-C₁₋₆alkyl-; heteroaryl-C₁₋₆alkyl-; andheterocyclyl-C₁₋₆alkyl-; wherein heteroaryl is a 5-6 membered ringhaving one, two, or three heteroatoms each independently selected fromN, O, or S; wherein heteroaryl is optionally substituted with one ormore substituents each independently selected from R^(b); whereinheterocyclyl is a 4-7 membered ring optionally substituted by one ormore substituents each independently selected from R^(c); wherein whenheterocyclyl contains a —NH— moiety, that —NH-moiety is optionallysubstituted by R^(d); wherein C₂₋₆alkenyl and C₂₋₆alkynyl, are eachindependently optionally substituted by one or more substituents eachindependently selected from R^(e); wherein C₁₋₆alkyl is optionallysubstituted by one or more substituents each independently selected fromR^(f); wherein C₃₋₆cycloalkyl is independently optionally substituted byone or more substituents each independently selected from R^(g).

In some cases, R² and R⁴ may be independently selected from the groupconsisting of hydrogen and —OR^(x), provided that at least one of R² andR⁴ is hydrogen, wherein R^(x) may be selected from the group consistingof hydrogen; halogen; C₁₋₆alkyl; C₂₋₆alkenyl; C₂₋₆alkynyl;C₃₋₆cycloalkyl; phenyl; naphthyl; heteroaryl; heterocyclyl;C₃₋₆cycloalkyl-C₁₋₆alkyl-; phenyl-C₁₋₆alkyl-; naphthyl-C₁₋₆alkyl-;heteroaryl-C₁₋₆alkyl-; and heterocyclyl-C₁₋₆alkyl-; wherein heteroarylis a 5-6 membered ring having one, two, or three heteroatoms eachindependently selected from N, O, or S; wherein heteroaryl is optionallysubstituted with one or more substituents each independently selectedfrom R^(b); wherein heterocyclyl is a 4-7 membered ring optionallysubstituted by one or more substituents each independently selected fromR^(c); wherein when heterocyclyl contains a —NH— moiety, that —NH—moiety is optionally substituted by R^(d); wherein C₂₋₆alkenyl andC₂₋₆alkynyl, are each independently optionally substituted by one ormore substituents each independently selected from R^(e); whereinC₁₋₆alkyl is optionally substituted by one or more substituents eachindependently selected from R^(f); wherein C₃₋₆cycloalkyl isindependently optionally substituted by one or more substituents eachindependently selected from R^(g);

R^(b) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; C₁₋₆alkyl;C₂₋₆alkenyl; C₂₋₆alkynyl; C₃₋₆cycloalkyl; C₁₋₆alkoxy; C₃₋₆alkenyloxy;C₃₋₆alkynyloxy; C₃₋₆cycloalkoxy; C₁₋₆alkyl-S(O)_(w)—, where w is 0, 1,or 2; C₁₋₆alkylC₃₋₆cycloalkyl-; C₃₋₆cycloalkyl-C₁₋₆alkyl-;C₁₋₆alkoxycarbonyl-N(R^(a))—; C₁₋₆alkylN(R^(a))—;C₁₋₆alkyl-N(R^(a))carbonyl-; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl-;R^(a)R^(a′)N-carbonyl-N(R^(a))—; R^(a)R^(a′)N—SO₂—; andC₁₋₆alkyl-carbonyl-N(R^(a))—;

R^(a) and R^(a′) may be selected, independently for each occurrence,from the group consisting of hydrogen and C₁₋₆alkyl, or R^(a) and R^(a′)when taken together with the nitrogen to which they are attached form a4-6 membered heterocyclic ring, wherein C₁₋₆alkyl is optionallysubstituted by one or more substituents each independently selected fromthe group consisting of halogen, oxo, and hydroxyl, and wherein theheterocyclic ring is optionally substituted by one or more substituentseach independently selected from the group consisting of halogen, alkyl,oxo, or hydroxyl;

R^(c) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; oxo; C₁₋₆alkyl;C₂₋₆alkenyl; C₂₋₆alkynyl; C₃₋₆cycloalkyl; C₁₋₆alkoxy; C₃₋₆alkenyloxy;C₃₋₆alkynyloxy; C₃₋₆cycloalkoxy; C₁₋₆alkyl-S(O)_(w)—, where w is 0, 1,or 2; C₁₋₆alkylC₃₋₆cycloalkyl-; C₃₋₆cycloalkyl-C₁₋₆alkyl-;C₁₋₆alkoxycarbonyl-N(R^(a))—; C₁₋₆alkylN(R^(a))—;C₁₋₆alkyl-N(R^(a))carbonyl-; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl-;R^(a)R^(a′)N-carbonyl-N(R^(a))—; R^(a)R^(a′)N—SO₂—; andC₁₋₆alkyl-carbonyl-N(R^(a))—;

R^(d) may be selected, independently for each occurrence, from the groupconsisting of C₁₋₆alkyl, C₁₋₆alkylcarbonyl, and C₁₋₆alkylsulfonyl,wherein C₁₋₆alkyl is optionally substituted by one or more substituentseach independently selected from halogen, hydroxyl, and R^(a)R^(a′)N—;

R^(e) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; C₁₋₄alkoxy;C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl;R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, where w is 0, 1, or 2;

R^(f) may be selected, independently for each occurrence, from the groupconsisting of halogen; hydroxyl; —NO₂; —N₃; —CN; —SCN; C₁₋₄alkoxy;C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl;R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, where w is 0, 1, or 2;

R^(g) may be selected, independently for each occurrence, from the groupconsisting of halogen, hydroxyl, —NO₂; —N₃; —CN; —SCN; C₁₋₆alkyl;C₁₋₄alkoxy; C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl;R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, where w is 0, 1, or 2.

In certain embodiments, R⁵ and R⁶ may be independently selected from thegroup consisting of -Q-Ar and hydrogen; wherein Q is independentlyselected from the group consisting of C₁₋₆alkyl; C₂₋₆alkenyl;C₂₋₆alkynyl; C₃₋₆cycloalkyl; heterocyclyl; C₃₋₆cycloalkyl-C₁₋₆alkyl-;heterocyclyl-C₁₋₆alkyl-; and a bond; and wherein Ar is selected from thegroup consisting substituted or unsubstituted phenyl, naphthyl, andheteroaryl; or R⁵ and R⁶, together with the atoms to which they areattached, form a 4-6 membered heterocyclic or cycloalkyl ring,optionally substituted by one or more substituents each independentlyselected from halogen, hydroxyl, —NO₂; —N₃; —CN; —SCN; C₁₋₆alkyl;C₁₋₄alkoxy; C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—; R^(a)R^(a′)N-carbonyl;R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, where w is 0, 1, or 2; and

wherein R^(a) and R^(a′) may be selected, independently for eachoccurrence, from the group consisting of hydrogen and C₁₋₆alkyl, orR^(a) and R^(a′) when taken together with the nitrogen to which they areattached form a 4-6 membered heterocyclic ring, wherein C₁₋₆alkyl isoptionally substituted by one or more substituents each independentlyselected from the group consisting of halogen, oxo, and hydroxyl, andwherein the heterocyclic ring is optionally substituted by one or moresubstituents each independently selected from the group consisting ofhalogen, alkyl, oxo, or hydroxyl.

In certain embodiments, at least one of R¹, R², R³, and R⁴ may behydroxyl.

In some instances, at least one of R¹, R², R³, and R⁴ may be C₁-C₆alkyl, optionally substituted with one, two, or three substituentsselected independently from the group consisting of halogen, hydroxyl,—NH₂, and cyano.

In some embodiments, at least one of R⁵ and R⁶ may be —(C₁-C₆alkylene)-Ar. At least one of R⁵ and R⁶ may also be —CH₂—Ar. In somecases, at least one of R⁵ and R⁶ is -Q-phenyl. In certain examples, oneof R⁵ and R⁶ may be hydrogen.

In some cases, R⁷ and R⁸ may be independently selected from the groupconsisting of hydrogen; halogen; hydroxyl; C₁-C₆ alkyl; phenyl; andnaphthyl; or R⁷ and R⁸, together with the atoms to which they areattached, form a 4-6 membered heterocyclic or cycloalkyl ring; whereinC₁-C₆ alkyl, phenyl, naphthyl, the cycloalkyl ring, and the heterocyclicring each may be substituted independently by one or more substituentsselected from the group consisting of halogen; hydroxyl; —NO₂; —N₃; —CN;—SCN; C₁₋₄alkoxy; C₁₋₄alkoxycarbonyl; R^(a)R^(a′)N—;R^(a)R^(a′)N-carbonyl; R^(a)R^(a′)N—SO₂—; and C₁₋₄alkylS(O)_(w)—, wherew is 0, 1, or 2; wherein R^(a) and R^(a′) may be selected, independentlyfor each occurrence, from the group consisting of hydrogen andC₁₋₆alkyl, or R^(a) and R^(a′) when taken together with the nitrogen towhich they are attached form a 4-6 membered heterocyclic ring, whereinC₁₋₆alkyl is optionally substituted by one or more substituents eachindependently selected from the group consisting of halogen, oxo, andhydroxyl, and wherein the heterocyclic ring is optionally substituted byone or more substituents each independently selected from the groupconsisting of halogen, alkyl, oxo, or hydroxyl.

In some cases, R⁷ and R⁸ may be hydrogen.

In an exemplary embodiment, a compound may be represented by:

In another exemplary embodiment, a compound may be represented by:

In yet another exemplary embodiment, a compound may be represented by:

For example, provided herein is a compound represented by:

wherein:

R¹, R², R³, and R⁴ are each independently selected from the groupconsisting of hydrogen; halogen, C₁-C₆alkyl, or —OH;

R⁵ is selected from the group consisting of —CH₂-phenyl and hydrogen,provided that R⁵ is —CH₂-phenyl when R¹ and R³ are —OH and R² and R⁴ aremethyl;

X is selected from the group consisting of OR^(x) and NR^(x)R^(x),wherein R^(x) is independently selected, for each occurrence, from thegroup consisting of hydrogen, and C₁-C₆alkyl; and pharmaceuticallyacceptable salts, stereoisomers, and hydrates thereof.

The compounds of the present disclosure and formulations thereof mayhave a plurality of chiral centers. Each chiral center may beindependently R, S, or any mixture of R and S. For example, in someembodiments, a chiral center may have an R:S ratio of between about100:0 and about 50:50, between about 100:0 and about 75:25, betweenabout 100:0 and about 85:15, between about 100:0 and about 90:10,between about 100:0 and about 95:5, between about 100:0 and about 98:2,between about 100:0 and about 99:1, between about 0:100 and 50:50,between about 0:100 and about 25:75, between about 0:100 and about15:85, between about 0:100 and about 10:90, between about 0:100 andabout 5:95, between about 0:100 and about 2:98, between about 0:100 andabout 1:99, between about 75:25 and 25:75, and about 50:50. Formulationsof the disclosed compounds comprising a greater ratio of one or moreisomers (i.e., R and/or S) may possess enhanced therapeuticcharacteristic relative to racemic formulations of a disclosed compoundsor mixture of compounds.

Disclosed compounds may provide for efficient cation channel opening atthe NMDA receptor, e.g. may bind or associate with the glutamate site ofthe NMDA receptor to assist in opening the cation channel. The disclosedcompounds may be used to regulate (turn on or turn off) the NMDAreceptor through action as an agonist.

The compounds as described herein may be glycine site NMDA receptorpartial agonists. A partial agonist as used in this context will beunderstood to mean that at a low concentration, the analog acts as anagonist and at a high concentration, the analog acts as an antagonist.Glycine binding is not inhibited by glutamate or by competitiveinhibitors of glutamate, and also does not bind at the same site asglutamate on the NMDA receptor. A second and separate binding site forglycine exists at the NMDA receptor. The ligand-gated ion channel of theNMDA receptor is, thus, under the control of at least these two distinctallosteric sites. Disclosed compounds may be capable of binding orassociating with the glycine binding site of the NMDA receptor. In someembodiments, disclosed compounds may possess a potency that is 10-foldor greater than the activity of existing NMDA receptor glycine sitepartial agonists. For example, disclosed compounds may possess a 10-foldto 20-fold enhanced potency compared to GLYX-13. GLYX-13 is representedby:

For example, provided herein are compounds that may be at least about20-fold more potent as compared to GLYX-13, as measured by burstactivated NMDA receptor-gated single neuron conductance (I_(NMDA)) in aculture of hippocampal CA1 pyramidal neurons at a concentration of 50nM. In another embodiment, a provided compound may be capable ofgenerating an enhanced single shock evoked NMDA receptor-gated singleneuron conductance (I_(NMDA)) in hippocampal CA1 pyramidal neurons atconcentrations of 100 nM to 1 μM. Disclosed compounds may have enhancedpotency as compared to GLYX-13 as measured by magnitude of long termpotentiation (LTP) at Schaffer collateral-CA-1 synapses in in vitrohippocampal slices.

The disclosed compounds may exhibit a high therapeutic index. Thetherapeutic index, as used herein, refers to the ratio of the dose thatproduces a toxicity in 50% of the population (i.e., TD₅₀) to the minimumeffective dose for 50% of the population (i.e., ED₅₀). Thus, thetherapeutic index=(TD₅₀):(ED₅₀). In some embodiments, a disclosedcompound may have a therapeutic index of at least about 10:1, at leastabout 50:1, at least about 100:1, at least about 200:1, at least about500:1, or at least about 1000:1.

Compositions

In other aspects, formulations and compositions comprising the disclosedcompounds and optionally a pharmaceutically acceptable excipient areprovided. In some embodiments, a contemplated formulation comprises aracemic mixture of one or more of the disclosed compounds.

Contemplated formulations may be prepared in any of a variety of formsfor use. By way of example, and not limitation, the compounds may beprepared in a formulation suitable for oral administration, subcutaneousinjection, or other methods for administering an active agent to ananimal known in the pharmaceutical arts.

Amounts of a disclosed compound as described herein in a formulation mayvary according to factors such as the disease state, age, sex, andweight of the individual. Dosage regimens may be adjusted to provide theoptimum therapeutic response. For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for themammalian subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier.

The specification for the dosage unit forms of the invention aredictated by and directly dependent on (a) the unique characteristics ofthe compound selected and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin.

The compounds can be administered in a time release formulation, forexample in a composition which includes a slow release polymer. Thecompounds can be prepared with carriers that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations aregenerally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating thecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In accordance with an alternative aspect of the invention, a compoundmay be formulated with one or more additional compounds that enhance thesolubility of the compound.

Methods

Methods for treating cognitive disorders and for enhancing learning areprovided. Such methods include administering a pharmaceuticallyacceptable formulation of one or more of the disclosed compounds to apatient in need thereof. Also contemplated are methods of treatingpatients suffering from, memory deficits associated with aging,schizophrenia, special learning disorders, seizures, post-strokeconvulsions, brain ischemia, hypoglycemia, cardiac arrest, epilepsy,migraine, as well as Huntington's, Parkinson's and Alzheimer's disease.

Other methods contemplated include the treatment of cerebral ischemia,stroke, brain trauma, brain tumors, acute neuropathic pain, chronicneuropathic pain, sleep disorders, drug addiction, depression, certainvision disorders, ethanol withdrawal, anxiety, memory and learningdisabilities, autism, epilepsy, AIDS dementia, multiple system atrophy,progressive supra-nuclear palsy, Friedrich's ataxia, Down's syndrome,fragile X syndrome, tuberous sclerosis, olivio-ponto-cerebellar atrophy,cerebral palsy, drug-induced optic neuritis, peripheral neuropathy,myelopathy, ischemic retinopathy, diabetic retinopathy, glaucoma,cardiac arrest, behavior disorders, impulse control disorders,Alzheimer's disease, memory loss that accompanies early stageAlzheimer's disease, attention deficit disorder, ADHD, schizophrenia,amelioration of opiate, nicotine addiction, ethanol addition, traumaticbrain injury, spinal cord injury, post-traumatic stress syndrome, andHuntington's chorea.

For example, provided herein is a method of treating depression in apatient need thereof, comprising administering a disclosed compound, e.gby acutely administering a disclosed compound. In certain embodiments,the treatment-resistant patient is identified as one who has beentreated with at least two types of antidepressant treatments prior toadministration of a disclosed compound. In other embodiments, thetreatment-resistant patient is one who is identified as unwilling orunable to tolerate a side effect of at least one type of antidepressanttreatment.

The most common depression conditions include Major Depressive Disorderand Dysthymic Disorder. Other depression conditions develop under uniquecircumstances. Such depression conditions include but are not limited toPsychotic depression, Postpartum depression, Seasonal affective disorder(SAD), mood disorder, depressions caused by chronic medical conditionssuch as cancer or chronic pain, chemotherapy, chronic stress, posttraumatic stress disorders, and Bipolar disorder (or manic depressivedisorder).

Refractory depression occurs in patients suffering from depression whoare resistant to standard pharmacological treatments, includingtricyclic antidepressants, MAOIs, SSRIs, and double and triple uptakeinhibitors and/or anxiolytic drugs, as well non-pharmacologicaltreatments such as psychotherapy, electroconvulsive therapy, vagus nervestimulation and/or transcranial magnetic stimulation. A treatmentresistant-patient may be identified as one who fails to experiencealleviation of one or more symptoms of depression (e.g., persistentanxious or sad feelings, feelings of helplessness, hopelessness,pessimism) despite undergoing one or more standard pharmacological ornon-pharmacological treatment. In certain embodiments, atreatment-resistant patient is one who fails to experience alleviationof one or more symptoms of depression despite undergoing treatment withtwo different antidepressant drugs. In other embodiments, atreatment-resistant patient is one who fails to experience alleviationof one or more symptoms of depression despite undergoing treatment withfour different antidepressant drugs. A treatment-resistant patient mayalso be identified as one who is unwilling or unable to tolerate theside effects of one or more standard pharmacological ornon-pharmacological treatment.

In yet another aspect, a method for enhancing pain relief and forproviding analgesia to an animal is provided.

In certain embodiments, methods for treating schizophrenia are provided.For example, paranoid type schizophrenia, disorganized typeschizophrenia (i.e., hebephrenic schizophrenia), catatonic typeschizophrenia, undifferentiated type schizophrenia, residual typeschizophrenia, post-schizophrenic depression, and simple schizophreniamay be treated using the methods and compositions contemplated herein.Psychotic disorders such as schizoaffective disorders, delusionaldisorders, brief psychotic disorders, shared psychotic disorders, andpsychotic disorders with delusions or hallucinations may also be treatedusing the compositions contemplated herein.

Paranoid schizophrenia may be characterized where delusions or auditoryhallucinations are present, but thought disorder, disorganized behavior,or affective flattening are not. Delusions may be persecutory and/orgrandiose, but in addition to these, other themes such as jealousy,religiosity, or somatization may also be present.

Disorganized type schizophrenia may be characterized where thoughtdisorder and flat affect are present together.

Catatonic type schizophrenia may be characterized where the subject maybe almost immobile or exhibit agitated, purposeless movement. Symptomscan include catatonic stupor and waxy flexibility.

Undifferentiated type schizophrenia may be characterized where psychoticsymptoms are present but the criteria for paranoid, disorganized, orcatatonic types have not been met.

Residual type schizophrenia may be characterized where positive symptomsare present at a low intensity only.

Post-schizophrenic depression may be characterized where a depressiveepisode arises in the aftermath of a schizophrenic illness where somelow-level schizophrenic symptoms may still be present.

Simple schizophrenia may be characterized by insidious and progressivedevelopment of prominent negative symptoms with no history of psychoticepisodes.

In some embodiments, methods are provided for treating psychoticsymptoms that may be present in other mental disorders, including, butnot limited to, bipolar disorder, borderline personality disorder, drugintoxication, and drug-induced psychosis.

In another embodiment, methods for treating delusions (e.g.,“non-bizarre”) that may be present in, for example, delusional disorderare provided.

Also provided are methods for treating social withdrawal in conditionsincluding, but not limited to, social anxiety disorder, avoidantpersonality disorder, and schizotypal personality disorder.

Additionally, methods are provided for treating obsessive-compulsivedisorder (OCD).

EXAMPLES

The following examples are provided for illustrative purposes only, andare not intended to limit the scope of the disclosure.

General Methods

All solvents used were of laboratory grade solvents. Tetrahydrofuran waspredistilled over KOH and then distilled over Na/benzophenone underargon. Dichloromethane was distilled over CaH2. Diisopropyl amine wasdistilled over KOH.

Column chromatography was conducted on silica gel 100-200 mesh. For TLCpurpose commercially available aluminum backed plates coated with silicagel 60 F254 from Merck, Darmstadt, West Germany were used.

NMR spectra were recorded on a Varian-Unity Inova 500 MHz, and BrukerAvance III 400 MHz instruments. All NMR spectra were determined indeuterated DMSO and chemical shifts are reported as δ values in ppm withtetramethylsilane was an internal standard (δ=0). Coupling constants (J)are given in Hertz. Signals in the 1H NMR spectra are characterized as s(silnglet), d (doublet), t (triplet), m (multiplet), and br s (broadsinglet).

Chemical purities were determined by UPLC on Waters Aquity system byusing either aq.TFA/aq.MeCN or aq.NH4OAc/aq.MeCN with a PDA detector.Mass were determined on Schimadzu 2010 EV LCMS system by using eitheraq.TFA/aq.MeCN or aq.NH4OAc/aq.MeCN with a PDA detector. Chiral puritieswere determined by using Chiralpak (IA) column (250×4.6 mm, 5 um) on aAgilent-1200 series using n-hexane:ethanol as mobile phase with PDAdetector.

Optical rotation were determined in chloroform and water in a 2-mL cellwith 50 mm path length on a JASCO P-2000 polarimeter.

Example 1—Synthesis of(S)—N-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-1-((S)-1-((S)-2-amino-3-hydroxypropanoyl)pyrrolidine-2-carbonyl)pyrrolidine-2-carboxamide(Compound A)

The following reaction sequence was used (Scheme A) to synthesize(S)—N-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-1-((S)-1-((S)-2-amino-3-hydroxypropanoyl)pyrrolidine-2-carbonyl)pyrrolidine-2-carboxamide

Synthesis of (S)-tert-butyl1-((S)-3-acetoxy-2-(benzyloxycarbonylamino)-propanoyl)-pyrrolidine-2-carboxylate(2)

(S)-3-Acetoxy-2-(benzyloxycarbonylamino)-propanoic acid (1.5 g, 5.33mmol) was dissolved in CH₂Cl₂ (15 mL). N-Methylmorpholine (NMM) (0.64mL, 5.87 mmol) and isobutyl chloroformate (IBCF) (0.72 mL, 6.12 mmol)were added at −15° C. and stirred for 30 minutes under inert atmosphere.A mixture of (S)-tert-butyl pyrrolidine-2-carboxylate (1) (998 mg, 5.87mmol) and NMM (0.64 mL, 5.87 mmol) in DMF (5 mL) were added drop wise tothe reaction mixture and stirring was continued for another 3 h at RT.The reaction mixture was diluted with DCM (200 mL), washed with water(50 mL), citric acid solution (10 mL) and brine (10 mL). The separatedorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The obtained crude residue was purified by silica gelcolumn chromatography eluting with 30% EtOAc/Hexane to afford compound 2(1.6 g, 69.5%).

¹H-NMR: (200 MHz, DMSO-d₆): δ 7.81-7.76 (d, J=20.5 Hz, 1H), 7.35-7.30(m, 5H), 5.03-4.97 (m, 2H), 4.61-4.55 (m, 1H), 4.32-4.16 (m, 2H),4.08-3.87 (m, 2H), 3.65-3.59 (m, 1H), 2.21-2.11 (m, 2H), 1.98 (s, 3H),1.91-1.75 (m, 2H), 1.37 (s, 9H).

Mass m/z: 435.0 [M⁺+1].

Synthesis of(S)-1-((S)-3-acetoxy-2-(benzyloxycarbonylamino)-propanoyl)-pyrrolidine-2-carboxylicacid (3)

To a solution of compound 2 (1 g, 2.30 mmol) in CH₂Cl₂ (5 mL) was added20% TFA-DCM (10 mL) and stirred at RT for 2 h. The reaction mixture wasdiluted with water (10 mL) and extracted with EtOAc (2×15 mL). Theorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to yield compound 3 (800 mg, 92%).

¹H-NMR: (200 MHz, DMSO-d₆): δ 12.58 (br s, 1H), 7.81-7.77 (d, J=8.0 Hz,1H), 7.35-7.27 (m, 5H), 5.04-4.96 (m, 2H), 4.66-4.60 (m, 1H), 4.32-4.24(m, 2H), 4.04-3.86 (m, 1H), 3.66-3.59 (t, J=12.6 Hz, 2H), 2.17-2.07 (m,3H), 1.98-1.80 (m, 4H).

Mass m/z: 379.0 [M⁺+1].

Synthesis of (2S,3R)-methyl2-((S)-1-((S)-1-((R)-3-acetoxy-2-(benzyloxycarbonylamino)-propanoyl)-pyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)-3-hydroxybutanoate(5)

Compound 3 (1.0 g, 2.64 mmol) was dissolved in CH₂Cl₂ (10 mL), NMM (0.32g, 3.17 mmol) and IBCF (0.41 g, 3.04 mmol) were added to the reactionmixture at −15° C. and stirred for 30 minutes under inert atmosphere. Amixture of (2S,3R)-methyl3-hydroxy-2-((S)-pyrrolidine-2-carboxamido)-butanoate (4) (0.73 g, 3.17mmol) and NMM (0.35 mL) in DMF (3 mL) were added drop wise to thereaction mixture at −15° C. and stirring was continued for another 3 hat RT. The reaction mixture was diluted with DCM (200 mL), washed withwater (20 mL), citric acid solution (2×20 mL) and brine (2×50 mL). Theseparated organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure. The crude residue obtained was purified bysilica gel column chromatography eluting with 5% CH₃OH/EtOAc to affordcompound (5) (0.29 g, 19%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.83-7.81 (m, 1H), 7.72-7.70 (m, 1H),7.36-7.35 (m, 5H), 5.07-5.01 (m, 2H), 4.99-4.93 (m, 1H), 4.58 (s, 1H),4.50-4.48 (m, 1H), 4.26-4.22 (m, 2H), 4.07-4.00 (m, 2H), 3.89-3.86 (m,1H), 3.61-3.55 (m, 5H), 3.53 (s, 1H), 3.39 (s, 1H), 2.12 (s, 1H), 1.98(s, 3H), 1.94-1.83 (m, 4H), 1.81-1.80 (m, 3H), 1.05 (d, J=6.5 Hz, 3H).

Mass m/z: 591.0 [M⁺+1].

Synthesis ofbenzyl-(R)-1-((S)-2-((S)-2-((2S,3R)-1-(aminooxy)-3-hydroxy-1-oxobutan-2-ylcarbamoyl)-pyrrolidine-1-carbonyl)-pyrrolidin-1-yl)-3-hydroxy-1-oxopropan-2-ylcarbamate(6)

A solution of methanolic ammonia (3 mL) was added to compound 5 (0.28 g,0.47 mmol) and stirred at RT for 18 h. The volatiles were evaporatedunder reduced pressure to afford compound 6 (0.21 g, 82.3%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.38-7.31 (m, 5H), 7.26 (s, 1H), 7.10-7.03(m, 2H), 6.65 (br s, 1H), 5.04-5.01 (m, 2H), 4.98-4.84 (m, 1H),4.76-4.75 (m, 1H), 4.61 (s, 1H), 4.38-4.31 (m, 2H), 4.02-4.00 (m, 2H),3.77-3.74 (m, 1H), 3.67-3.56 (m, 3H), 3.44-3.37 (m, 2H), 2.14-1.86 (m,8H), 1.01-1.00 (m, 3H).

Mass m/z: 550 [M⁺+1].

Synthesis of(S)—N-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-1-((S)-1-((S)-2-amino-3-hydroxypropanoyl)-pyrrolidine-2-carbonyl)-pyrrolidine-2-carboxamide(Compound A)

To a solution of compound 6 (0.21 g, 0.39 mmol) in methanol (5 mL) wasadded 10% Pd/C (30 mg) and the reaction mixture was stirred underhydrogen atmosphere for 2 h. The reaction mixture was filtered overcelite, solvent was evaporated in vacuo, and the crude residue obtainedwas triturated with diethyl ether to yield A (130 mg, 83.3%).

¹H-NMR: (500 MHz, DMSO-d₆) (Rotamers): δ 7.39 (d, J=8.0 Hz, 1H),7.08-7.03 (m, 2H), 6.65 (br s, 1H), 4.89-4.85 (m, 1H), 1.61-1.59 (m,1H), 4.39-4.38 (m, 1H), 4.02-4.00 (m, 2H), 3.68-3.52 (m, 4H), 3.43-3.36(m, 2H), 3.22-3.10 (m, 2H), 2.19-2.13 (m, 1H), 2.07-1.98 (m, 1H),1.93-1.81 (m, 5H), 1.75 (s, 2H), 1.01-1.00 (m, 3H).

LCMS m/z: 400.2 [M⁺+1].

HPLC Purity: 99.27%.

Synthesis of (S)-1-(benzyloxycarbonyl) pyrrolidine-2-carboxylic acid (8)

To a stirred solution of (S)-pyrrolidine-2-carboxylic acid (7) (2.0 g,17.39 mmol) in THF:H₂O (20 mL, 1:1) were added Na₂CO₃ (2.76 g, 26.08mmol) and Cbz-Cl (3.54 g, 20.80 mmol) and stirred at RT for 18 h. Thereaction mixture was washed with EtOAc (10 mL) and the aqueous layer wasacidified with 3N HCl and extracted with EtOAc (2×20 mL). The combinedorganic layer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to yield compound 8 (3.0 g, 69.7%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.62 (br s, 1H), 7.36-7.22 (m, 5H),5.12-5.00 (m, 2H), 4.24-4.15 (dd, J=5.0, 36.0 Hz, 1H), 3.46-3.31 (m,2H), 2.25-2.15 (m, 1H), 1.94-1.79 (m, 3H).

Mass m/z: 250.0 [M⁺+1].

Synthesis of (S)-benzyl2-((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-ylcarbamoyl)pyrrolidine-1-carboxylate(9)

Compound 8 (5.0 g, 20.08 mmol) was dissolved in CH₂Cl₂ (50 mL), NMM(2.43 mL, 22.08 mmol) and IBCF (2.74 mL, 23.09 mmol) were added andstirred at −15° C. for 30 minutes under inert atmosphere. A mixture of(2S,3R)-methyl 2-amino-3-hydroxybutanoate (2.93 g, 22.08 mmol) and NMM(2.43 mL, 22.08 mmol) in DMF (15 mL) were added drop wise at −15° C. Theresultant reaction mixture was stirred at RT for 3 h. It was dilutedwith DCM (200 mL) and the organic layer was washed with water (50 mL),brine (50 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The obtained crude was purified by silica gel columnchromatography eluting with 30% EtOAc/Hexane to afford compound 9 (3.1g, 42%).

¹H-NMR: (500 MHz, DMSO-d₆)(Rotamers): δ 7.98-7.94 (m, 1H), 7.35-7.27 (m,5H), 5.09-4.94 (m, 3H), 4.44 (dd, J=5.5, 8.5 Hz, 1H), 4.29-4.27 (m, 1H),4.12 (s, 1H), 3.62 (s, 3H), 3.44-3.30 (m, 2H), 2.20-2.08 (m, 1H),1.87-1.78 (m, 3H), 1.08-0.94 (2d, 3H).

Mass m/z: 365.0 [M⁺+1].

Example 2—Synthesis of(S)—N—((S)-1-amino-3-hydroxy-1-oxopropan-2-yl)-1-((S)-1-((2S,3R)-2-amino-3-hydroxybutanoyl)pyrrolidine-2-carbonyl) pyrrolidine-2-carboxamide (Compound B)

The following reaction sequence was used (Scheme B) to synthesize(S)—N—((S)-1-amino-3-hydroxy-1-oxopropan-2-yl)-1-((S)-1-((2S,3R)-2-amino-3-hydroxybutanoyl)pyrrolidine-2-carbonyl) pyrrolidine-2-carboxamide:

Synthesis of (S)-1-(tert-butoxycarbonyl)-pyrrolidine-2-carboxylic acid(2)

To an ice cold stirred solution of (S)-pyrrolidine-2-carboxylic acid (1)(3.0 g, 26.08 mmol) in THF:H₂O (60 mL, 1:1) were added Na₂CO₃ (5.52 g,52.16 mmol), Boc₂O (6.25 g, 26.69 mmol) and stirred at RT for 16 h. Thereaction mixture was diluted with water and washed with EtOAc (50 mL).The aqueous layer was acidified with 2N HCl and extracted with EtOAc(2×100 mL). The combined organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure to yield the(S)-1-(tert-butoxycarbonyl)-pyrrolidine-2-carboxylic acid (2) (4.8 g,86%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.49 (br s, 1H), 4.08-4.03 (m, 1H),3.36-3.24 (m, 2H), 2.22-2.11 (m, 1H), 1.87-1.76 (m, 3H), 1.39 (s, 9H).

Mass m/z: 216.0 [M⁺+1].

Synthesis of (S)-tert-butyl2-((S)-3-hydroxy-1-methoxy-1-oxopropan-2-ylcarbamoyl)-pyrrolidine-1-carboxylate(3)

Compound 2 (2.0 g, 9.00 mmol) was dissolved in CH₂Cl₂ (10 mL) cooled to−15° C., NMM (1.12 mL, 10.2 mmol) and IBCF (1.26 mL, 1.15 mmol) wereadded and stirred at 0° C. for 20 minutes. A mixture of (S)-methyl2-amino-3-hydroxypropanoate (1.59 g, 10.2 mmol) and NMM (1.12 mL) in DMF(3 mL) were added drop wise at −15° C. and the resultant reactionmixture was stirred at RT for 1 h. It was diluted with DCM (200 mL),water (50 mL) and washed with 2N HCl (20 mL) and brine (2×50 mL). Theseparated organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure. The crude residue obtained was purified bysilica gel column chromatography eluting with 20% EtOAc/Hexane to affordcompound 3 (2.3 g) as a syrup.

Mass m/z: 317.0 [M⁺+1].

Synthesis of (S)-methyl 3-hydroxy-2-((S)-pyrrolidine-2-carboxamido)propionate (4)

(S)-Tert-butyl-2-((S)-3-hydroxy-1-methoxy-1-oxopropan-2-ylcarbamoyl)-pyrrolidine-1-carboxylate(3) (500 mg, 1.58 mmol) was dissolved in 1,4-dioxane (3 mL) and a HClsolution in dioxane (3.16 mL, 3.16 mmol) was added stirred at RT for 4h. The volatiles were evaporated under reduced pressure to affordcompound 4 (280 mg) as solid.

¹H-NMR: (200 MHz, DMSO-d₆): δ 9.99 (br s, 1H), 9.12-9.08 (m, 1H), 8.53(br s, 1H), 5.48 (br s, 2H), 4.43-4.22 (m, 2H), 3.82-3.67 (m, 4H), 3.56(s, 3H), 2.36-2.27 (m, 1H), 1.93-1.86 (m, 3H).

Mass m/z: 217.0 [M⁺+1].

Synthesis of (S)-methyl 2-((S)-1-((S)-1-((2R,3S)-3-acetoxy-2-(benzyloxycarbonylamino)-butanoyl)-pyrrolidine-2-carbonyl)-pyrrolidine-2-carboxamido)-3-hydroxypropanoate(6)

(2S)-1-((2R)-3-acetoxy-2-(benzyloxycarbonylamino)-butanoyl)-pyrrolidine-2-carboxylicacid (5) (1.3 g, 2.62 mmol) was dissolved in CH₂Cl₂ (15 mL), NMM (0.43mL) and IBCF (0.51 mL) was added at −10° C. and stirred for 30 minutesunder inert atmosphere. A mixture of(S)-methyl-3-hydroxy-2-((S)-pyrrolidine-2-carboxamido)-propionate (4)(992 mg, 3.93 mmol) and NMM (0.43 mL) in DMF (5 mL) were added drop wiseto the reaction mixture and stirring was continued for another 3 h atRT. The reaction mixture was diluted with DCM (200 mL), washed withwater (20 mL), citric acid solution (2×20 mL) and brine (2×50 mL). Theseparated organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure. The obtained crude material was purified bysilica gel column chromatography eluting with 5% CH₃OH/CH₂Cl₂ to affordcompound 6 (270 mg, 17.5%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.13 (d, J=8.0 Hz, 1H), 7.74 (d, J=7.5 Hz,1H), 7.38-7.31 (m, 5H), 5.08-4.96 (m, 3H), 4.85-4.82 (m, 1H), 4.56 (d,J=8.0 Hz, 1H), 4.44-4.42 (m, 2H), 4.27 (d, J=7.0 Hz, 1H), 4.10 (d,J=10.5 Hz, 2H), 3.81-3.78 (m, 1H), 3.72-3.70 (m, 1H), 3.61-3.59 (m, 3H),3.54-3.50 (m, 2H), 2.16-2.14 (m, 1H), 2.05-2.01 (m, 1H), 1.90 (s, 3H),1.87-1.86 (m, 3H), 1.85-1.84 (m, 3H), 1.21-1.20 (d, J=6.0 Hz, 3H).

Mass m/z: 591.0 [M⁺+1].

Synthesis ofBenzyl-(2R,3S)-1-((S)-2-((S)-2-((S)-1-(aminooxy)-3-hydroxy-1-oxopropan-2-ylcarbamoyl)pyrrolidine-1-carbonyl)pyrrolidin-1-yl)-3-hydroxy-1-oxobutan-2-ylcarbamate(7)

To a solution of compound 6 (250 g, 0.42 mmol) in CH₃OH (2 mL) was addedMeOH—NH₃ (10 mL) and was stirred at RT for 16 h. The volatiles wereevaporated under reduced pressure to afford compound 7 (190 mg, 84%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.60 (d, J=7.5 Hz, 1H), 7.35-7.30 (m, 5H),7.18 (d, J=7.0 Hz, 1H), 7.11-7.06 (m, 2H), 5.05-4.97 (m, 2H), 4.82-4.81(m, 1H), 4.60-4.59 (m, 2H), 4.33-4.31 (m, 1H), 4.15-4.08 (m, 2H),3.81-3.79 (m, 1H), 3.72-3.64 (m, 2H), 3.59-3.53 (m, 4H), 2.14 (s, 1H),2.03 (d, J=9.0 Hz, 1H), 1.95-1.85 (m, 5H), 1.75 (s, 1H), 1.10 (d, J=6.5Hz, 3H).

Mass m/z: 550.0 [M⁺+1].

Synthesis of(S)—N—((S)-1-amino-3-hydroxy-1-oxopropan-2-yl)-1-((S)-1-((2S,3R)-2-amino-3-hydroxybutanoyl)pyrrolidine-2-carbonyl)-pyrrolidine-2-carboxamide (B):

To a solution of compound 7 (190 mg, 0.35 mmol) in methanol (5 mL) wasadded 10% Pd/C (50 mg) and the reaction mixture was stirred underhydrogen atmosphere for 2 h. The reaction mixture was filtered through acelite pad, solvent was evaporated in vacuo and the crude was purifiedby column chromatography on basic alumina using 0-5% CH₃OH in CH₂Cl₂ aseluent to yield compound B (130 mg, 73%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.65-7.60 (m, 1H), 7.12-7.03 (m, 2H), 4.81(br s, 1H), 4.58-4.57 (m, 1H), 4.49 (m, 1H), 4.38-4.19 (m, 1H),4.10-4.06 (m, 1H), 3.69-3.62 (m, 2H), 3.59-3.56 (m, 4H), 3.49-3.45 (m,2H), 3.37-3.26 (m, 2H), 2.19-2.15 (m, 1H), 2.09-1.99 (m, 1H), 1.95-1.84(m, 5H), 1.75 (s, 1H), 1.06 (d, J=13.0 Hz, 3H).

LCMS m/z: 400.8 [M⁺+1].

HPLC Purity: 97.71%.

Example 3—Synthesis of(S)—N—((S)-1-amino-3-hydroxy-1-oxopropan-2-yl)-1-((S)-1-((S)-2-amino-3-hydroxy-propanoyl)pyrrolidine-2-carbon-pyrrolidine-2-carboxamide(Compound C)

The following reaction sequence was used (Scheme C) to synthesize(S)—N—((S)-1-amino-3-hydroxy-1-oxopropan-2-yl)-1-((S)-1-((S)-2-amino-3-hydroxy-propanoyl)-pyrrolidine-2-carbonyl)-pyrrolidine-2-carboxamide

Synthesis of (S)-1-(tert-butoxycarbonyl)-pyrrolidine-2-carboxylic acid(2)

To a stirred solution of (S)-pyrrolidine-2-carboxylic acid (3.0 g, 26.08mmol) in THF:H₂O (60 mL, 1:1) at 0° C. were added Na₂CO₃ (5.52 g, 52.16mmol) and Boc₂O (6.25 g, 26.69 mmol) and stirred at RT for 16 h. Thereaction mixture was diluted with water and washed with EtOAc (50 mL).The aqueous layer was acidified with 2N HCl and extracted with EtOAc(2×50 mL). The combined organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure to yield the(S)-1-(tert-butoxycarbonyl)-pyrrolidine-2-carboxylic acid 2 (4.8 g,85.7%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.49 (br s, 1H), 4.08-4.03 (m, 1H),3.36-3.24 (m, 2H), 2.22-2.11 (m, 1H), 1.87-1.76 (m, 3H), 1.39 (s, 9H).

Mass m/z: 216.0 [M⁺+1].

Synthesis of (S)-tert-butyl2-((S)-3-hydroxy-1-methoxy-1-oxopropan-2-ylcarbamoyl)pyrrolidine-1-carboxylate (3)

Compound 2 (2.0 g, 9.00 mmol) was dissolved in CH₂Cl₂ (10 mL) cooled to−15° C., NMM (1.12 mL, 10.2 mmol) and IBCF (1.26 mL, 1.15 mmol) wereadded and stirred at 0° C. for 20 minutes. A mixture of(S)-methyl-2-amino-3-hydroxypropanoate (1.59 g, 10.2 mmol) and NMM (1.12mL) in DMF (3 mL) were added drop wise at −15° C. The resultant reactionmixture was stirred at RT for 1 h. The reaction mixture was diluted withDCM (200 mL) and water (25 mL) and was washed with 2N HCl (20 mL) andbrine (10 mL). The separated organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The obtained crudematerial was purified by silica gel column chromatography eluting with20% EtOAc/Hexane to afford compound 3 (2.3 g) as solid.

Mass m/z: 317.0 [M⁺+1].

Synthesis of (S)-methyl 3-hydroxy-2-((S)-pyrrolidine-2-carboxamido)propanoate (4)

To a solution of(S)-tert-butyl-2-((S)-3-hydroxy-1-methoxy-1-oxopropan-2-ylcarbamoyl)pyrrolidine-1-carboxylate 3 (500 mg, 1.58 mmol) in 1,4-dioxane (3 mL)was added a solution of HCl in dioxane (3.16 mL, 3.16 mmol) and stirredat RT for 4 h. The volatiles were evaporated under reduced pressure toafford compound 4 (280 mg) as solid.

¹H-NMR: (200 MHz, DMSO-d₆): δ 9.99 (br s, 1H), 9.12-9.08 (m, 1H), 8.53(br s, 1H), 5.48 (br s, 2H), 4.43-4.22 (m, 2H), 3.82-3.67 (m, 4H), 3.56(s, 3H), 2.36-2.27 (m, 1H), 1.93-1.86 (m, 3H).

Mass m/z: 217.0 [M⁺+1].

Synthesis of (S)-methyl2-((S)-1-((S)-1-((S)-2-(benzyloxycarbonylamino)-3-hydroxypropanoyl)-pyrrolidine-2-carbonyl)-pyrrolidine-2-carboxamido)-3-hydroxypropanoate(6)

(S)-1-((S)-3-Acetoxy-2-(benzyloxycarbonylamino)-propanoyl)-pyrrolidine-2-carboxylicacid (5) (400 mg, 1.05 mmol) was dissolved in CH₂Cl₂ (2 mL), NMM (0.13mL) and IBCF (0.14 mL) were added at −15° C. and stirred for 30 minutesunder inert atmosphere. A mixture of(S)-methyl-3-hydroxy-2-((S)-pyrrolidine-2-carboxamido)-propanoatehydrochloride (4) (293 mg, 1.16 mmol) and NMM (0.13 mL) in DMF (2 mL)were added drop wise to the reaction mixture and stirring was continuedfor another 3 h at RT. The reaction mixture was diluted with DCM (200mL), washed with water (20 mL) and brine (10 mL). The separated organiclayer was dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. The obtained crude material was purified by silica gel columnchromatography eluting with 5% CH₃OH/CH₂Cl₂ to afford compound 6 (80 mg,13%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.09 (d, J=7.5 Hz, 1H), 7.71 (d, J=8.0 Hz,1H), 7.36-7.31 (m, 6H), 5.07-4.99 (m, 3H), 4.59-4.58 (m, 2H), 4.41-4.40(m, 1H), 4.29-4.24 (m, 3H), 3.86 (t, J=9.5 Hz, 1H), 3.72-3.68 (m, 1H),3.64-3.57 (m, 3H), 3.40-3.38 (m, 3H), 2.14-2.01 (m, 2H), 1.98 (s, 3H),1.90-1.80 (m, 6H).

Mass m/z: 535.0 [M⁺+1].

Synthesis ofBenzyl-(S)-1-((S)-2-((S)-2-((S)-1-amino-3-hydroxy-1-oxopropan-2-ylcarbamoyl)pyrrolidine-1-carbonyl)pyrrolidin-1-yl)-3-hydroxy-1-oxopropan-2-ylcarbamate (7)

To a solution of(S)-methyl-2-((S)-1-((S)-1-((S)-2-(benzyloxycarbonylamino)-3-hydroxypropanoyl)-pyrrolidine-2-carbonyl)-pyrrolidine-2-carboxamido)-3-hydroxypropanoate(6) (60 mg, 1.04 mmol) in MeOH was added MeOH—NH₃ (3 mL) was stirred atRT for 16 h. The volatiles were evaporated under reduced pressure toafford compound 7 (30 mg, 55%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.60 (d, J=7.5 Hz, 1H), 7.36-7.31 (m, 6H),7.11-7.06 (m, 2H), 5.04-4.98 (m, 2H), 4.82-4.74 (m, 2H), 4.61-4.59 (m,1H), 4.36-4.30 (m, 2H), 4.10-4.07 (m, 1H), 3.67-3.65 (m, 2H), 3.59-3.55(m, 6H), 3.44-3.40 (m, 2H), 1.95-1.92 (m, 6H).

Mass m/z: 520.0 [M⁺+1].

Synthesis of(S)—N—((S)-1-amino-3-hydroxy-1-oxopropan-2-yl)-1-((S)-1-((S)-2-amino-3-hydroxy-propanoyl)-pyrrolidine-2-carbonyl)-pyrrolidine-2-carboxamide(C)

Benzyl-(S)-1-((S)-2-((S)-2-((S)-1-amino-3-hydroxy-1-oxopropan-2-ylcarbamoyl)pyrrolidine-1-carbonyl)pyrrolidin-1-yl)-3-hydroxy-1-oxopropan-2-ylcarbamate 7 (300 mg, 0.57mmol) was dissolved in methanol (8 mL), 10% Pd/C (50 mg) was added andreaction mixture was stirred under hydrogen atmosphere for 2 h. Thereaction mixture was filtered and the filtrate was concentrated underreduced pressure to yield compound C (150 mg, 68%).

¹H-NMR: (500 MHz, DMSO-d₆) (Rotamers): δ 7.62 (d, J=8.0 Hz, 1H), 7.24(br s, 1H), 7.14-7.07 (m, 2H), 4.87-4.82 (m, 2H), 4.59-4.57 (m, 1H),4.37-4.31 (m, 2H), 4.11-4.07 (m, 2H), 3.70-3.39 (m, 8H), 2.17-2.01 (m,2H), 1.95-1.79 (m, 6H).

LCMS m/z: 386.4 [M⁺+1].

HPLC Purity: 98.45%.

Example 4—Synthesis ofN-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-1-((S)-1-((2S,3R)-2-amino-3-hydroxybutanoyl)-pyrrolidine-2-carbonyl)-2-benzylpyrrolidine-2-carboxamide(Compound D & E)

The following reaction sequence was used (Scheme D) to synthesizeN-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-1-((S)-1-((2S,3R)-2-amino-3-hydroxybutanoyl)-pyrrolidine-2-carbonyl)-2-benzsaylpyrrolidine-2-carboxamide(Compound D & E):

Synthesis of 1-Benzyl 2-ethyl 2-benzylpyrrolidine-1,2-dicarboxylate (2)

To a solution of (S)-1-benzyl-2-ethyl-pyrrolidine-1,2-dicarboxylate (1)(10 g, 36.10 mmol) in THF (150 mL) under inert atmosphere was addedLiHMDS (1M in THF) (43.3 mL, 43.3 mmol) at −25° C. and stirred for 2 h.Benzyl bromide (5.17 mL, 43.26 mmol) was added drop wise at −25° C. tothe reaction mixture. It was allowed to warm to RT and stirred for 2 h.The reaction mixture was cooled to 5° C., quenched with saturated NH₄Clsolution and the aqueous layer was extracted with EtOAc (2×200 mL). Thecombined organic extracts were dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The crude residue obtained waspurified by silica gel column chromatography eluting with 5%EtOAc/hexane to afford compound 2 (13 g, 75%) as liquid.

¹H-NMR: (200 MHz, DMSO-d₆): δ 7.47-7.32 (m, 5H), 7.27-7.16 (m, 3H),7.07-7.04 (m, 2H), 5.29-5.06 (m, 2H), 4.16-3.89 (m, 2H), 3.57-3.33 (m,2H), 3.02-2.78 (m, 2H), 2.13-1.89 (m, 2H), 1.56-1.51 (m, 1H), 1.21-1.04(m, 3H), 0.93-0.79 (m, 1H).

Mass m/z: 368.2 [M⁺+1].

Synthesis of 2-benzyl-1-(benzyloxycarbonyl)-pyrrolidine-2-carboxylicacid (3)

To a stirred solution of compound 2 (8.0 g, 21.79 mmol) in CH₃OH (20 mL)was added 2N aqueous KOH (20 mL) and heated up to 100° C. and stirredfor 16 h. The volatiles were evaporated under reduced pressure. Theresidue obtained was diluted with ice cold water (50 mL) and washed withether (50 mL). The aqueous layer was acidified to pH-2 using HClsolution and extracted with EtOAc (2×100 mL). The combined organic layerwas dried over anhydrous Na₂SO₄ and concentrated under reduced pressureto afford compound 3 (6 g, 81%) as an off white solid.

¹H-NMR: (200 MHz, DMSO-d₆): δ 12.71 (br s, 1H), 7.40-7.30 (m, 5H),7.25-7.19 (m, 3H), 7.07-7.00 (m, 2H), 5.27-5.02 (m, 2H), 3.59-3.32 (m,2H), 3.02-2.83 (m, 2H), 2.13-1.91 (m, 2H), 1.58-1.49 (m, 1H), 0.90-0.77(m, 1H).

Mass m/z: 340.1 [M⁺+1].

Synthesis ofBenzyl-2-benzyl-2-((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-ylcarbamoyl)-pyrrolidine-1-carboxylate(4)

To a suspension of compound 3 (1.0 g, 2.94 mmol), L-threonine methylester (471 mg, 3.53 mmol) in DMF (20 mL) was added HATU (1.12 g, 2.94mmol) and DIPEA (1.58 mL, 8.84 mmol) at 5° C. The reaction mixture wasstirred at RT for 16 h. It was diluted with EtOAc (150 mL) and washedwith water (2×30 mL). The organic layer was washed with brine, driedover Na₂SO₄, concentrated and purified by silica gel columnchromatography 50% EtOAc/Hexane as eluent to yield compound 4 (1.0 g,74%).

¹H-NMR: (200 MHz, DMSO-d₆): δ 7.62-7.59 (m, 1H), 7.44-7.31 (m, 5H),7.21-7.18 (m, 3H), 7.06-6.99 (m, 2H), 5.25-5.24 (m, 1H), 5.12-4.94 (m,2H), 4.30 (s, 1H), 4.15-4.08 (m, 1H), 3.66-3.64 (m, 3H), 3.63-3.49 (m,2H), 3.14 (s, 1H), 2.89 (s, 1H), 2.09-2.02 (m, 2H), 1.56-1.51 (m, 1H),1.09-0.98 (m, 4H).

Mass m/z: 455.1 [M⁺+1], 477.3 [M+Na].

Synthesis ofBenzyl-2-((2S,3R)-3-acetoxy-1-methoxy-1-oxobutan-2-ylcarbamoyl)-2-benzylpyrrolidine-1-carboxylate(5):

Compound 4 (3 g, 6.60 mmol) was dissolved in THF (30 mL), Et₃N (1.11 mL,7.92 mmol) and Ac₂O (742 mg, 7.26 mmol) were added at RT. The reactionmixture was stirred at RT for 2 h. The volatiles were evaporated underreduced pressure and the residue obtained was diluted with CH₂Cl₂ andwashed with dilute HCl. The combined organic extracts were dried overNa₂SO₄ and concentrated under reduced pressure. The crude residue waspurified by column chromatography using 30% EtOAc/Hexane as eluent toyield compound 5 (2.5 g, 76%).

¹H-NMR: (500 MHz, DMSO-d₆) (Rotamers): δ 8.15-7.71 (m, 1H), 7.42-7.04(m, 10H), 5.30-5.19 (m, 2H), 5.11-5.09 (m, 1H), 4.99-4.93 (m, 1H),4.67-4.62 (m, 1H), 3.66-3.64 (m, 3H), 3.55-3.46 (m, 2H), 3.38-3.35 (m,1H), 2.88-2.69 (m, 1H), 2.17-2.00 (m, 2H), 1.98-1.92 (m, 3H), 1.56-1.46(m, 1H), 1.23-1.17 (m, 3H), 1.02-0.86 (m, 1H).

LCMS m/z: 497.4 [M⁺+1].

Synthesis of (2S,3R)-methyl3-acetoxy-2-(2-benzylpyrrolidine-2-carboxamido)-butanoate (6)

To a stirring solution of compound 5 (4 g, 8.06 mmol) in ethanol (50 mL)was added 10% Pd/C (1.2 g) and the reaction mixture was stirred under H₂atmosphere (balloon pressure) for 4 h. It was filtered through celitepad and the filtrate was concentrated under reduced pressure to yieldcompound 6 (2.2 g, 75%).

¹H-NMR: (500 MHz, DMSO-d₆) (Rotamers): δ 8.22-8.17 (m, 1H), 7.24-7.16(m, 5H), 5.17 (t, J=11.5 Hz, 1H), 4.48-4.42 (m, 1H), 3.60-3.54 (s, 3H),3.20 (t, J=13.5 Hz, 1H), 3.06-2.97 (m, 1H), 2.82-2.68 (m, 3H), 2.08-2.02(m, 1H), 1.89 (s, 3H), 1.72-1.51 (m, 3H), 1.10 (2d, 3H).

LCMS m/z: 363 [M⁺+1], 385 [M+Na].

Synthesis of (S)-benzyl2-(2-((2S,3R)-3-acetoxy-1-methoxy-1-oxobutan-2-ylcarbamoyl)-2-benzylpyrrolidine-1-carbonyl)pyrrolidine-1-carboxylate (7)

To a stirred solution of compound 6 (1 g, 2.76 mmol) and Na₂CO₃ (732 mg,6.90 mmol) in CH₂Cl₂:H₂O (20 mL, 1:1) was added a solution of acidchloride [To a solution of (S)-1-(benzyloxycarbonyl)pyrrolidine-2-carboxylic acid (825 mg, 3.31 mmol) in CH₂Cl₂ (20 mL) wasadded SOCl₂ (0.60 mL) drop wise at 0° C. and was refluxed for 2 h. Thevolatiles were removed under reduced pressure to yield (S)-benzyl2-(chlorocarbonyl) pyrrolidine-1-carboxylate] in CH₂Cl₂ and the reactionmixture was stirred at RT for 2 h. The volatiles were evaporated underreduced pressure. The residue was diluted with CH₂Cl₂ (100 mL), filteredand the filtrate was concentrated under vacuum. The crude residue waspurified by column chromatography using 60% EtOAc/Hexane as eluent toafford compound 7 (750 mg, 45%).

¹H-NMR: (500 MHz, DMSO-d₆) (Rotamers): δ 7.36-7.23 (m, 8H), 7.15-7.12(m, 3H), 5.21-5.15 (m, 2H), 5.04-4.92 (m, 1H), 4.57-4.50 (m, 2H), 3.88(d, J=14.5 Hz, 1H), 3.65 (s, 3H), 3.54-3.46 (m, 3H), 3.21-3.13 (m, 1H),3.02-2.90 (m, 2H), 2.19-2.02 (m, 4H), 1.97 (s, 3H), 1.89 (s, 1H),1.77-1.65 (m, 1H), 1.17 (s, 2H), 1.06 (s, 2H).

Mass m/z: 594.1 [M⁺+1].

Synthesis of (2S,3R)-methyl3-acetoxy-2-(2-benzyl-1-((S)-pyrrolidine-2-carbonyl)-pyrrolidine-2-carboxamido)butanoate (8)

To a solution of compound 7 (200 mg, 0.336 mmol) in EtOAc (15 mL) wasadded 10% Pd/C (40 mg) was added under inert atmosphere and stirred for12 h under H₂ atmosphere (balloon pressure). The reaction mixture wasfiltered through celite pad and concentrated under reduced pressure. Theobtained residue was triturated with ether (10 mL) to afford compound 8(125 mg, 81%) as solid.

¹H-NMR: (500 MHz, CDCl₃) (Rotamers): δ 7.88-7.87 (d, 1H, J=8.5),7.30-7.26 (m, 2H), 7.24-7.21 (m, 1H), 7.13-7.12 (d, 2H, J=7), 5.44-5.43(m, 1H), 4.76-4.74 (m, 1H), 3.94-3.92 (m, 1H), 3.84-3.81 (m, 1H), 3.75(s, 3H), 3.50 (m, 1H), 3.26-3.12 (m, 3H), 2.90-2.88 (m, 1H), 2.23-2.15(m, 4H), 2.04 (s, 3H), 1.87-1.77 (m, 5H), 1.27-1.24 (m, 3H).

Mass m/z: 460(M+1).

Synthesis of Benzyl-2-(tert-butoxycarbonylamino)-3-hydroxybutanoate (10)

To a solution of 2-(tert-butoxycarbonylamino)-3-hydroxybutanoic acid(3.0 g, 13.69 mmol) in DMF (50 mL) was added K₂CO₃ (3.73 g, 27.39 mmol)and stirred at RT for 15 min. (Bromomethyl)benzene (2.81 g, 16.43 mmol)was added and stirred at RT for 6 h. The reaction mixture was dilutedwith water (50 mL) and extracted with EtOAc (2×50 mL). The combinedorganic layer was washed with brine (50 mL), dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The crude material was purifiedby silica gel column chromatography using 20% EtOAc/hexane as eluent toafford benzyl 2-(tert-butoxycarbonylamino)-3-hydroxybutanoate 10 (2.8 g,66%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.37-7.30 (m, 5H), 6.60 (d, J=8.5 Hz, 1H),5.18-5.08 (m, 2H), 4.76 (d, J=7 Hz, 1H), 4.08-4.00 (m, 2H), 1.38 (s,9H), 1.09 (d, J=6.0 Hz, 3H).

Mass m/z: 310.0 [M⁺+1], 210 [M⁺-De Boc].

Synthesis of benzyl-3-acetoxy-2-(tert-butoxycarbonylamino)-butanoate(11)

To a stirred solution ofbenzyl-2-(tert-butoxycarbonylamino)-3-hydroxybutanoate (2.8 g, 9.06mmol) in THF (80 mL) was added Ac₂O (1.1 g, 10.87 mmol), Et₃N (1.51 mL,10.87 mmol) and DMAP (280 mg) and stirred at RT for 15 min. Thevolatiles were removed under reduced pressure. The residue obtained wasdiluted with EtOAc (150 mL) and washed with cold 0.5N HCl solution (2×20mL). The organic layer was washed with brine, dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to afford3-acetoxy-2-(tert-butoxycarbonylamino)-butanoate 11 (2.8 g, 88%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.35-7.34 (m, 5H), 7.27-7.25 (d, J=8.5 Hz,1H), 5.18-5.06 (m, 3H), 4.34-4.32 (m, 1H), 1.90 (s, 3H), 1.39 (s, 9H),1.16 (d, J=3 Hz, 3H).

Mass m/z: 252 [M⁺+1-De Boc].

Synthesis of (2S,3R)-3-acetoxy-2-(tert-butoxycarbonylamino)-butanoicacid (12)

Benzyl-3-acetoxy-2-(tert-butoxycarbonylamino) butanoate 11 (1.4 g, 3.98mmol) was dissolved in EtOAc (40 mL), 10% Pd/C (600 mg) was added andreaction mixture was stirred under hydrogen atmosphere for 16 h. Thereaction mixture was filtered over celite, solvent was evaporated invacuo and the crude residue was triturated with hexane to yield(2S,3R)-3-acetoxy-2-(tert-butoxycarbonylamino) butanoic acid 12 (0.7 g,70%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.78 (br s, 1H), 6.94 (d, J=9.5 Hz, 1H),5.16-5.14 (m, 1H), 4.17-4.15 (m, 1H), 1.95 (s, 3H), 1.39 (s, 9H), 1.10(d, J=6.0 Hz, 3H).

Mass m/z: 260.0 [M−1].

Synthesis of(2S,3R)-methyl-3-acetoxy-2-(1-((S)-1-((2S,3R)-3-acetoxy-2-(tert-butoxycarbonyl-amino)-butanoyl)-pyrrolidine-2-carbonyl)-2-benzylpyrrolidine-2-carboxamido)-butanoate(13)

To a solution of compound(2S,3R)-3-acetoxy-2-(tert-butoxycarbonylamino)-butanoic acid 12 (199 mg,0.76 mmol) in CH₂Cl₂ (6 mL) was under inert atmosphere were added IBCF(125 mg, 0.91 mmol) and NMM (154 mg, 1.52 mmol) at −15° C. and stirredfor 1 h. A solution of (2S,3R)-methyl3-acetoxy-2-(2-benzyl-1-((S)-pyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)-butanoate 8 (350 mg, 0.76 mmol) in DMF (2 mL)was added to the reaction mixture and stirred for 1 h at −15° C. Theresultant reaction mixture was allowed to warm to RT and stirred for 19h. The reaction mixture was extracted with EtOAc and the separatedorganic layer was washed with water (20 mL), followed by brine (20 mL),dried over Na₂SO₄ and concentrated under reduced pressure. The crudematerial was purified by preparative HPLC to afford compound 13 (100 mg,20%).

¹H-NMR: (500 MHz, CD₃OD) (Rotamers): δ 7.30-7.24 (m, 3H), 7.15-7.13 (m,2H), 4.62-4.55 (m, 2H), 4.29-3.97 (m, 1H), 3.98-3.79 (m, 4H), 3.75 (s,3H), 3.62-3.22 (m, 2H), 3.23 (d, J=13.5 Hz, 1H), 3.00-2.95 (q, 1H),2.37-2.31 (m, 1H), 2.23-2.10 (m, 2H), 2.02-1.88 (m, 3H), 1.46-1.28 (m,2H), 0.97 (d, J=7.0 Hz, 6H).

Synthesis ofN-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-1-((S)-1-((2S,3R)-2-amino-3-hydroxybutanoyl)-pyrrolidine-2-carbonyl)-2-benzylpyrrolidine-2-carboxamide(D & E)

A solution of compound 13 (100 mg, 0.153 mmol) in methanolic-NH₃ (10 mL)was stirred in a sealed tube at RT for 72 h. The reaction mixture wasconcentrated under reduced pressure. The obtained crude residue waswashed with ether (2×2 mL) to afford a diastereomeric mixture ofCompound D & E (85 mg). 85 mg of this mixture was further purified bychiral preparative HPLC to yield 15 mg each of Compound D and E.

¹H-NMR: (500 MHz, CD₃OD) (Rotamers): δ 7.33-7.26 (m, 3H), 7.16 (s, 2H),4.55-4.54 (m, 1H), 4.39 (s, 1H), 4.14 (s, 1H), 4.01-3.98 (m, 1H),3.91-3.71 (m, 3H), 3.59 (s, 2H), 3.25-3.16 (m, 1H), 3.04-3.00 (m, 1H),2.33-2.10 (m, 3H), 2.01-1.91 (m, 2H), 1.86-1.80 (m, 1H), 1.46-1.44 (m,1H), 1.34-1.29 (m, 1H), 1.25-1.19 (m, 3H), 0.99-0.97 (d, J=14.0 Hz, 3H).

Mass m/z: 503 [M+].

HPLC Purity: 98.1%.

Example 5—[³H] MK-801 Binding Assay

This example demonstrates a [³H] MK-801 binding assay that may be usedto assess agonistic and/or antagonistic properties of candidate NMDAreceptor modulators.

Crude synaptic membranes were prepared from rat forebrains as describedin Moskal et al. (2001), “The use of antibody engineering to createnovel drugs that target N-methyl-D-aspartate receptors,” Curr. DrugTargets, 2:331-45. Male 2-3 month old rats were decapitated withoutanesthesia by guillotine, and the brains were rapidly removed (˜90 sec)and whole cortex and hippocampus dissected on an ice cold platform,frozen on dry ice, and stored at ˜80° C. Samples were homogenized in 20volumes of ice cold 5 mM Tris-HCl pH 7.4 by Brinkman Polytron andpelleted 48,000×g for 20 min at 4° C., and washed an additional 3 timesas described above. Membranes were then resuspended in 5 mM EDTA and 15mM Tris-HCl pH 7.4 and incubated for 1 hr at 37° C., membranes pelletedat 48,000×g for 20 min at 4° C., snap frozen in liquid nitrogen, andstored at −80° C. On the day of the experiment, membranes were thawed atroom temperature and washed an additional 7 times in ice cold 5 mMTris-HCl (pH 7.4) as described above. After the last wash, membraneswere resuspended in assay buffer (5 mM Tris-acetate pH 7.4), and proteincontent was determined by the BCA assay.

[³H] MK-801 binding assays were preformed as described in Urwyler et al.(2009), “Drug design, in vitro pharmacology, and structure-activityrelationships of 3-acylamino-2-aminopropionic acid derivatives, a novelclass of partial agonists at the glycine site on theN-methyl-D-aspartate (NMDA) receptor complex,” J. Med. Chem.,52:5093-10. Membrane protein (200 μg) was incubated with varyingconcentrations of the test compounds (10⁻³-10⁻¹⁷ M) with 50 μM glutamatefor 15 min at 23° C. Assay tubes were then incubated undernon-equilibrium conditions with [³H]MK-801 (5 nM; 22.5 Ci/mmol) for 15min at 23° C. followed by filtration through Whatman GF/B filters usinga Brandel M-24R Cell Harvester. Then the tubes were washed three timeswith assay buffer (5 mM Tris-acetate PH 7.4), and the filters wereanalyzed by liquid scintillation to calculate the disintegrations perminute (DPM). Zero levels were determined in the absence of any glycineligand and in the presence of 30 μM 5,7-Dichlorokynurenic acid(5,7-DCKA). Maximal stimulation was measured in the presence of 1 mMglycine. 50 VM glutamate was present in all samples.

For each data point (i.e., a single concentration of the test compound),the % maximal [³H] MK-801 binding was calculated by the followingformula:

% maximal [³H]MK-801binding=((DPM_((test compound))−DPM_(5,7-DCKA))/(DPM_(1 mM glycine)−DPM_(5,7-DCKA)))×100%

The efficacy for each compound, expressed as the % increase in [³H]MK-801 binding, is calculated by fitting the data to a “log(agonist) vs.response (three parameters)” equation using Graph Pad Prism, with theefficacy for the test compound being the best-fit top value.

TABLE 1 [³H] MK-801 Binding Assay Data. Efficacy Compound Potency (%Increase in [³H] MK-801 Binding) A   5 pM 79% B   6 pM 24% C  16 pM 23%D 0.2 pM 12% E 0.2 pM 12%

Example 6—NMDA Receptor (NMDAR) Currents

This example demonstrates an assay for determining the effect of testcompounds on NMDAR currents.

Experiments were conducted on hippocampal slices from 14-18 day oldSprague-Dawley rats as described in Zhang et al. (2008) “A NMDA receptorglycine site partial agonist, GLYX-13, simultaneously enhances LTP andreduces LTD at Schaffer collateral-CA1 synapses in hippocampus,”Neuropharmacology, 55:1238-50. Whole cell recordings were obtained fromCA1 pyramidal neurons voltage clamped at −60 mV, in slices perfused with(artificial cerebrospinal fluid) ACSF containing 0 mM [Mg2+] and 3 mM[Ca2+], plus 10 μM bicuculline and 20 μM CNQX to pharmacologicallyisolate NMDAR-dependent excitatory postsynaptic currents (EPSCs).Varying concentrations of test compound (10 nM to 1 μM) were bathapplied and Schaffer collateral fibers were stimulated with singleelectrical pulses (80 s duration) once every 30 s. NMDAR EPSCs werecharacterized by long rise and decay times, and were fully blocked atthe end of each experiment by bath application of the NMDAR-specificantagonist D-2-amino-5-phosphonopentanoic acid (D-AP5; 50 μM). Theefficacy of a test compound was calculated as the % increased in NMDARcurrent from the baseline. The baseline was measured as the NMDARcurrent before the test compound was applied.

TABLE 2 NMDAR Current Assay Data. Efficacy (% Change in NMDAR CurrentCompound Concentration from Baseline) A 1 μM 70% B NT NT C NT NT D 1 μM75% E 1 μM 10% NT = not tested.

Example 7—Long-Term Potentiation (LTP) Assay

This example demonstrates an assay for determining the effect of testcompounds on LTP.

Hippocampal slices from 14-18 day old Sprague-Dawley rats weretransferred to an interface recording chamber and continuously perfusedat 3 ml/min with oxygenated ACSF at 32±0.5° C. Low resistance recordingelectrodes were made from thin-walled borosilicate glass (1-2 MΩ afterfilling with ACSF) and inserted into the apical dendritic region of theSchaffer collateral termination field in stratum radiatum of the CA1region to record field excitatory postsynaptic potentials (fEPSPs). Abipolar stainless steel stimulating electrode (FHC Co.) was placed onSchaffer collateral-commissural fibers in CA3 stratum radiatum, andconstant current stimulus intensity adjusted to evoke approximatelyhalf-maximal fEPSPs once each 30 s (50-100 pA; 100 ms duration). fEPSPslope was measured by linear interpolation from 20%-80% of maximumnegative deflection, and slopes confirmed to be stable to within ±10%for at least 10 min before commencing an experiment. Long-termpotentiation (LTP) was induced by a high frequency stimulus train (3×100Hz/500 ms; arrow) at Schaffer collateral-CA1 synapses in control(vehicle), untreated slices, or slices pre-treated with test compound(10 nM to 100 μM). Long-term potentiation signals were recorded using aMulticlamp 700B amplifier and digitized with a Digidata 1322 (AxonInstruments, Foster City, Calif.). Data were analyzed using pClampsoftware (version 9, Axon Instruments) on an IBM-compatible personalcomputer. The efficacy was calculated as the % increase in long-termpotentiation measured for slices pre-treated with test compound ascompared to vehicle.

TABLE 3 LTP Assay Data. Compound Concentration Efficacy (% Increase fromVehicle) A NT NT B NT NT C NT NT D 1 uM 30% E 1 uM 10% NT = not tested.

Example 8—Porsolt Test

This example demonstrates the Porsolt test for assessing test compoundsfor antidepressant activity.

Experiments were conducted as described in Burgdorf et al. (2009) “Theeffect of selective breeding for differential rates of 50-kHz ultrasonicvocalizations on emotional behavior in rats,” Devel. Psychobiol.,51:34-46. Male Sprague-Dawley rats (2-3 month old) were dosed with testcompound (0.3 to 30 mg/kg; intravenously via tail vein injection, or peros via gastric gavage) or vehicle (1 ml/kg sterile saline, or 1 ml/kgDMSO for 2,5-diazaspiro[3.4]octan-1-one) in a blind manner 1 hr beforetesting. Animals were placed in a 46 cm tall×20 cm in diameter clearglass tube filled to 30 cm with tap water at room temperature (23°C.±0.5° C.) for 5 min on the test day. All animals were towel driedafter each swimming session by the experimenter. Water was changed afterevery other animal. Animals were videotaped and total duration (sec) offloating behavior (as defined as the minimal movement required in orderto maintain the animal's head above the water) was quantified by a blindexperimenter.

TABLE 4 Porsolt Assay Data. Compound Dose, Route % Reduction in FloatingA 3 mg/kg, i.v. 90% B NT NT C NT NT D 1 mg/kg, p.o. 84% E 1 mg/kg, p.o.63% NT = not tested.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications,websites, and other references cited herein are hereby expresslyincorporated herein in their entireties by reference.

What is claimed is:
 1. A compound represented by:

wherein: R¹, R², R³, and R⁴ are each independently selected from thegroup consisting of hydrogen; halogen, C₁-C₆alkyl, or —OH; R⁵ isselected from the group consisting of —CH₂-phenyl and hydrogen, providedthat R⁵ is —CH₂-phenyl when R¹ and R³ are —OH and R² and R⁴ are methyl;X is selected from the group consisting of OR^(x) and NR^(x)R^(x),wherein R^(x) is independently selected, for each occurrence, from thegroup consisting of hydrogen, and C₁-C₆alkyl; pharmaceuticallyacceptable salts, stereoisomers, and hydrates thereof.
 2. The compoundof claim 1 represented by:


3. The compound of claim 1 represented by:


4. The compound of claim 1 represented by:


5. The compound of claim 1 represented by:


6. The compound of claim 1 represented by:


7. The compound of claim 1 represented by:


8. The compound of claim 1 represented by:

wherein X is OH or NH₂.
 9. A pharmaceutical composition, comprising: atherapeutically effective amount of a compound of claim 1 and apharmaceutically acceptable carrier.
 10. The pharmaceutical compositionof claim 9, suitable for oral administration.
 11. The pharmaceuticalcomposition of claim 9, suitable for injection.
 12. A method of treatingdepression, Alzheimer's disease, attention deficit disorder, ADHD,schizophrenia, or anxiety, in a patient in need thereof, comprisingadministering to said patient: a pharmaceutically effective amount of acompound of claim 1.